Biological Macromolecules: Bioactivity

and Biomedical Applications Amit

Kumar Nayak
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BIOLOGICAL MACROMOLECULES
 BIOLOGICAL
MACROMOLECULES
BIOACTIVITY AND BIOMEDICAL
APPLICATIONS

Edited by

Amit Kumar Nayak

Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, India

Amal Kumar Dhara

Department of Pharmacy, Contai Polytechnic, Contai, India

Dilipkumar Pal
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur, India
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 Contents

List of contributors xiii 2.2.2 Lysostaphin 28

2.2.3 Metallo-β-lactamase-like lactonase 32
Preface xix 2.3 Chitosan as a bioactive polysaccharide 36
2.3.1 Relationship of chitosan physicochemical
property and its bioactivity 37
I 2.3.2 Bioactivity of chitosan with modified
Background functional group 41
2.4 Conclusion 43
References 43
1. Biological macromolecules: sources,
properties, and functions 3. The importance of biological
AMAL KUMAR DHARA AND AMIT KUMAR NAYAK macromolecules in biomedicine
AHMED OLATUNDE, OMAR BAHATTAB, ABDUR RAUF,
1.1 Introduction 3 NAVEED MUHAMMAD, YAHYA S. AL-AWTHAN,
1.2 Carbohydrates 4 TABUSSAM TUFAIL, MUHAMMAD IMRAN AND
MOHAMMAD S. MUBARAK
1.2.1 Monosaccharides 5
1.2.2 Oligosaccharides 5
3.1 Introduction 53
1.2.3 Polysaccharides 5
3.2 Biological macromolecules in biomedicine and
1.3 Lipids 9
therapies 53
1.3.1 Simple lipids 10
3.3 Carbohydrates 54
1.3.2 Compound or conjugate lipids 10
3.3.1 Therapeutics based on carbohydrates 55
1.3.3 Derived lipids 10
3.4 Peptides 56
1.4 Proteins 11
3.4.1 Therapeutics based on peptides 57
1.4.1 Simple proteins 12
3.5 Proteins 58
1.4.2 Conjugated proteins 13
3.5.1 Therapeutics based on proteins (proteins
1.4.3 Derived proteins 13
and monoclonal antibodies) 58
1.5 Nucleic acids 14
3.6 Lipids 60
1.5.1 Nucleotides 15
3.6.1 Drug delivery-based on lipids 60
1.5.2 Nucleosides 15
3.7 Nucleic acids and oligonucleotides 61
1.5.3 DNA 15
3.7.1 Therapeutics based on oligonucleotides 61
1.5.4 RNA 16
3.8 Synthesis of macromolecules 63
1.6 Conclusion 18
3.9 Biomedicine 64
References 18
3.10 Conclusions 65
References 65
2. Structure activity relationship of
biological macromolecules
4. Modification techniques for
AURELIE SARAH MOK TSZE CHUNG, YONG KIAT TEO,
WAI TENG CHENG AND JOASH BAN LEE TAN carbohydrate macromolecules
AJAY VASUDEO RANE, DEEPTI YADAV
2.1 Introduction 23 AND KRISHNAN KANNY
2.2 Enzymes as bioactive proteins 24
2.2.1 L-amino acid oxidases 25 4.1 Introduction 69

v
vi Contents

4.2 Cellulose 69 5.8.1 Polyunsaturated fatty acids 120

4.3 Hemicelluloses 71 5.8.2 The role of Omega-3 PUFAs in some
4.4 Lignin 72 disorders 121
4.5 Chitin and chitosan 72 5.9 The potential use of bioactive lipids in cancer stem
4.6 Modification of carbohydrate biological cells and coronavirus disease (COVID-19) 123
macromolecules 73 5.9.1 Bioactive lipids in cancer 123
References 86 5.9.2 Bioactive lipids in COVID-19 123
5.10 Carbohydrates as nutraceuticals 124
5.10.1 Brief overview of carbohydrates 124
II 5.10.2 Role of polysaccharides in extracellular
membrane 124
Bioactivity 5.10.3 Immunostimmulatory effect of
carbohydrates 125
5. Biological macromolecules as 5.10.4 Carbohydrates from plants with
nutraceutical activity 125
nutraceuticals
5.10.5 Cellulose and hemicellulose 125
IRERI ALEJANDRA CARBAJAL-VALENZUELA, 5.10.6 Animal derived carbohydrates with
NUVIA MARINA APOLONIO HERNANDEZ,
DIANA VANESA GUTIERREZ-CHAVEZ,
nutraceutical activity 126
BEATRIZ GONZÁLEZ-ARIAS, 5.10.7 Heparin 126
ALEJANDRA JIMENEZ-HERNANDEZ, 5.10.8 Hyaluronic acid 127
IRINEO TORRES-PACHECO, ENRIQUE RICO-GARCÍA, 5.10.9 Chitosan and chitin 127
ANA ANGELICA FEREGRINO-PÉREZ
5.10.10 Carbohydrates with nutraceutical activity
AND RAMÓN GERARDO GUEVARA-GONZÁLEZ
from microorganisms 128
5.1 History of the applications of nutraceutical 5.10.11 Alginate 128
compounds in health care 97 5.10.12 Dextran 128
5.2 Alkaloids 99 5.10.13 Bacillus striatum polysaccharide 128
5.2.1 Caffeine 99 5.11 Credit 129
5.2.2 Capsaicin 101 References 129
5.2.3 Theobromine 101
5.3 Phenolic compounds 102 6. Biological macromolecules as
5.3.1 Curcumin 102 antioxidants
5.3.2 Resveratrol 103 T. MADHUJITH, N.E. WEDAMULLA AND D.A.S. GAMAGE
5.3.3 Quercetin 103
5.3.4 Anthocyanins 103 6.1 Introduction 139
5.3.5 Luteolin 104 6.2 Types and sources of biological
5.3.6 Naringenin 104 macromolecules 141
5.3.7 Catechins 104 6.2.1 Polysaccharides 141
5.4 Terpenes 105 6.2.2 Proteins 149
5.4.1 Lycopene 105 6.2.3 Other antioxidative macromolecules 152
5.4.2 β-Carotene 105 6.3 Macromolecules as antioxidants 152
5.4.3 Lutein 106 6.3.1 Polysaccharides as antioxidants 152
5.4.4 Zeaxanthin 107 6.3.2 Proteins as antioxidants 155
5.5 Future views 107 6.3.3 Nonextractable polyphenols as
5.6 Proteins and peptides with biological activity of antioxidants 156
medical interest 107 6.4 Applications 156
5.7 Nucleic acids and their nutraceutical properties 6.4.1 Food-based applications 156
used in biomedicine 112 6.4.2 Other applications 158
5.7.1 Nucleic acids overview 112 6.5 Limitations of biological macromolecules 158
5.7.2 Perspectives 119 6.6 Future trends 159
5.8 Introduction of lipids 119 References 159
 Contents vii
7. Biological macromolecules as 8.2.1 Terpenoids 203
antimicrobial agents 8.2.2 Steroids 206
8.2.3 Phenolics 207
MD. SHAHRUZZAMAN, SHAFIUL HOSSAIN,
TANVIR AHMED, SUMAYA F. KABIR, 8.2.4 Alkaloids 208
MD. MINHAJUL ISLAM, ASHIQUR RAHMAN, 8.2.5 Polysaccharides 208
MD. SAZEDUL ISLAM, SABRINA SULTANA AND 8.2.6 Peptides 209
MOHAMMED MIZANUR RAHMAN 8.2.7 Polyketide 210
8.2.8 Polyunsaturated fatty acids 210
7.1 Introduction 165
8.3 Conclusion 213
7.2 Classification of biological macromolecule 167
References 213
7.2.1 Carbohydrate 167
7.2.2 Protein 169
7.2.3 Lipid 170 9. Biological macromolecules acting on
7.2.4 Nucleic acid 171
7.3 Antimicrobial activity of biological
central nervous system
macromolecules 172 DILIPKUMAR PAL AND KHUSHBOO RAJ

7.3.1 Polysaccharides 172

9.1 Introduction 219
7.3.2 Proteins 175
9.1.1 Proteins 219
7.3.3 Fatty acids 177
9.1.2 Cell cycle proteins 220
7.4 Antimicrobial activity of macromolecule
9.1.3 Homer/vesl proteins 220
composites 180
9.1.4 Central fatty hypothesis 222
7.4.1 Chitosan-alginate 180
9.1.5 Carbohydrates 222
7.4.2 Gelatin-chitosan 181
9.1.6 Role of carbohydrates on nervous
7.4.3 Keratin-chitosan 182
system 223
7.4.4 Collagen-alginate 183
9.1.7 In sensory organs 223
7.4.5 Chitosan-cellulose 184
9.1.8 Glycans 223
7.4.6 Lactoferrin-Oleic Acid 185
9.1.9 Role of glycan in neural development 224
7.5 Nanotechnology based antimicrobial
9.1.10 Lipids 224
macromolecule 185
9.1.11 Role of cPLA2 in cerebral ischemia 225
7.5.1 Chitosan based nanocomposite 185
9.1.12 In the case of neurodegenerative
7.5.2 Alginate-based nanocomposite 187
diseases 225
7.5.3 Cellulose based nanocomposite 187
9.1.13 Lipid peroxidation 225
7.5.4 Gelatin based nanocomposite 189
9.2 Conclusion 226
7.5.5 Collagen based nanocomposite 190
References 226
7.5.6 Keratin-based nanoparticle 190
7.5.7 Oleic acid based nanoparticle 190
7.6 Applications 190
7.6.1 Food packaging 190
10. Biological macromolecules as
7.6.2 Drug delivery 191 antidiabetic agents
7.6.3 Wound dressing 192 JAISON JEEVANANDAM, CALEB ACQUAH
7.7 Conclusion 193 AND MICHAEL K. DANQUAH

References 193
10.1 Introduction 229
8. Biological macromolecules from algae 10.2 Types of biological macromolecules 230
and their antimicrobial applications 10.3 Biological macromolecules 232
10.3.1 Carbohydrates 232
NATANAMURUGARAJ GOVINDAN,
GAANTY PRAGAS MANIAM, MOHD HASBI AB. RAHIM,
10.3.2 Lipids 233
AHMAD ZIAD SULAIMAN AND AZILAH AJIT 10.3.3 Proteins 235
10.3.4 Nucleic acids 237
8.1 Introduction 203 10.4 Advantages, limitations, and future
8.2 Bioactive macromolecules 203 perspectives 238
viii Contents

10.5 Conclusion 238 14. Synthetic macromolecules with

References 239
11. Biological macromolecules as
anticancer agents
HIMJA TIWARI, HARSHAL DESHMUKH,
NILESH SHIRISH WAGH AND JAYA LAKKAKULA
11.1 Introduction 243
11.2 Biological macromolecules for cancer therapy
11.2.1 Carbohydrates 244
11.2.2 Proteins and nucleic acid 258
11.2.3 Lipids 262
11.3 Conclusion 269
References 269
12. Biological macromolecules as
immunomodulators
EDUARDO COSTA, MANUELA MACHADO,
MANUELA PINTADO AND SARA SILVA
12.1 Introduction 273
12.2 Immunomodulation 274
12.3 Immunomodulation, biomolecules, and
applications 274
12.4 Polysaccharides 275
12.4.1 Immunomodulatory polysaccharides 275
12.4.2 Gut microbiota modulation 277
12.5 Lipids 277
12.5.1 Immunomodulatory effect of lipids 278
12.6 Proteins 280
12.6.1 Known immunomodulatory proteins 280
Acknowledgments 282
References 282
13. Biological macromolecules acting on
gastrointestinal systems
DILIPKUMAR PAL AND SUPRIYO SAHA
13.1 Introduction 289
13.2 Role of carbohydrates in gastrointestinal
system 289
13.3 Role of proteins in gastrointestinal system 295
13.4 Role of fatty acids in gastrointestinal system 298
13.5 Role of nucleic acids in gastrointestinal
system 300
13.6 Conclusion 301
References 302
biological activity
STEFANIA RACOVITA, MARCEL POPA,
LEONARD IONUT ATANASE AND SILVIA VASILIU
14.1 Introduction 305
14.2 Synthetic macromolecules with antimicrobial
activity 306
14.2.1 History of antimicrobial agents and
244 antimicrobial polymers 307
14.2.2 Classification of antimicrobial
polymers 308
14.2.3 Preparation routes for antimicrobial
polymers 309
14.2.4 Factors affecting the antimicrobial
activity 311
14.2.5 Synthetic macromolecules with
antibacterial activity 313
14.2.6 Synthetic macromolecules with antiviral
activity 317
14.2.7 Synthetic macromolecules with antifungal
activity 318
14.2.8 Synthetic macromolecules with
antiparasitic activity 319
14.3 Synthetic macromolecules with antioxidant
activity 320
14.4 Polymer sequestrants 324
14.5 Conclusions 328
References 328
Further reading 335
III
Functional applications
15. Biological macromolecules in drug
delivery
AMIT KUMAR NAYAK, MD SAQUIB HASNAIN,
ANINDITA BEHERA, AMAL KUMAR DHARA AND
DILIPKUMAR PAL
15.1 Introduction 339
15.2 Drug delivery using various biological
macromolecules 340
15.2.1 Drug delivery using carbohydrates 341
15.2.2 Drug delivery using proteins and
peptides 349
15.2.3 Drug delivery using nucleic acids 355
 Contents ix
15.2.4 Drug delivery using lipids 359 18.4 Biological macromolecules for delivery systems of
15.3 Conclusion 367 growth factors 424
References 367 18.4.1 Protein-based materials for growth factor
delivery 425
16. Biological macromolecules in tissue 18.4.2 Polysaccharide-based materials for growth
engineering factor delivery 427
18.4.3 Polysaccharide combinations for growth
PANDURANG APPANA DALAVI,
SESHA SUBRAMANIAN MURUGAN, factor delivery 429
SUKUMARAN ANIL AND JAYACHANDRAN VENKATESAN 18.4.4 Composites materials for growth factor
delivery 430
16.1 Introduction 381 18.4.5 Protein-based composite for growth factor
16.2 Bone tissue engineering 381 delivery 430
16.3 Biological macromolecules in bone tissue 18.4.6 Protein-polysaccharide composites for
engineering 382 growth factor delivery 432
16.3.1 Alginate 382 18.4.7 Polysaccharide-polysaccharide composites
16.3.2 Chitosan 383 for growth factor delivery 433
16.3.3 Carrageenan 384 References 433
16.3.4 Fucoidan 385
16.3.5 Ulvan 385 19. Biological macromolecules for growth
16.3.6 Gelatin 386 factor delivery in bone regeneration
16.4 Conclusion 387
ARISTEIDIS PAPAGIANNOPOULOS AND ELENI VLASSI
Acknowledgment 387
References 387 19.1 Introduction 439
19.2 Bone regeneration 439
17. Biological macromolecules for drug 19.3 Growth factors in tissue and bone
delivery in tissue engineering regeneration 441
MARCEL POPA AND LEONARD IONUT ATANASE 19.4 Biomacromolecules as carriers of growth
factors 443
17.1 Introduction 393 19.5 Hydrogels and sponges 445
17.2 Drug-loaded electrospun fibers used in tissue 19.6 Scaffolds and fibers 446
engineering applications and drug delivery 394 19.7 Nanoparticles and nanoassemblies 448
17.2.1 Drug-loaded polysaccharides-based 19.8 Concluding remarks 449
electrospun fibers 395 References 449
17.2.2 Drug-loaded protein-based electrospun
fibers 401 20. Biological macromolecules for
17.3 Drug-loaded injectable hydrogels used in tissue nutrients delivery
engineering applications and drug delivery 403
LONG CHEN, ZHONGYU YANG, DAVID JULIAN
17.4 Conclusions 411 MCCLEMENTS, ZHENGYU JIN AND MING MIAO
References 411
20.1 Introduction 455
18. Biological macromolecules for growth 20.2 Nutrients 455
factor delivery 20.2.1 Water-soluble nutrients 456
M.D. FIGUEROA-PIZANO 20.2.2 Oil-soluble nutrients 458
20.3 Biological macromolecules used for nutrients
18.1 Introduction 419 delivery 458
18.2 Delivery systems for growth factors 421 20.3.1 Polysaccharides 459
18.3 Materials for delivery systems of growth 20.3.2 Proteins 461
factors 423 20.3.3 Glycoproteins and proteoglycans 461
x Contents

20.3.4 Others (lignin as example) 462 22.2.4 Collagen 500

20.4 Molecular interactions that maintain the stability 22.2.5 Gelatin 503
of biopolymer-based delivery systems 462 22.2.6 Fibrin 504
20.4.1 Electrostatic interactions 463 22.2.7 Glycosaminoglycans 505
20.4.2 Hydrogen bonding 463 22.2.8 Silk (fibroion and spidroin) 506
20.4.3 Hydrophobic interactions 463 22.2.9 Other natural polymers in TE 508
20.4.4 Covalent interactions 464 22.3 Advantages, drawbacks, applications, forms and
20.5 Retention and release mechanisms 464 manufacturing methods 511
20.6 Nutrient delivery systems based on biological 22.4 Conclusions 526
macromolecules 465 Acknowledgment 527
20.6.1 Composition and structure 465 References 527
20.6.2 Fabrication 466
20.6.3 Properties 469 23. Biological macromolecules for enzyme
20.6.4 Applications 469 immobilization
20.7 Future trends 471
HAMZA RAFEEQ, SARMAD AHMAD QAMAR,
20.7.1 Co-encapsulation of multiple HIRA MUNIR, MUHAMMAD BILAL AND HAFIZ M.N. IQBAL
nutrients 471
20.7.2 Targeted and controlled release of 23.1 Introduction 529
bioactive molecules 471 23.2 Biological macromolecules for enzyme
20.7.3 In vivo testing 472 immobilization 532
References 472 23.2.1 Chitin and chitosan 532
23.2.2 Agarose 533
21. Biological macromolecules for nucleic 23.2.3 Alginate 534
acid delivery 23.2.4 Cellulose and its derivatives 535
AHMED S. ABO DENA AND IBRAHIM M. EL-SHERBINY
23.2.5 Gelatin for enzyme immobilization 536
23.2.6 Dextran for enzyme immobilization 537
21.1 Introduction 479 23.2.7 Pectin for enzyme immobilization 539
21.2 Nucleic acids structure and functions 480 23.2.8 Xanthan for enzyme immobilization 540
21.3 Biological macromolecules for nucleic acid 23.3 Conclusions and future outlook 541
delivery 482 Acknowledgment 541
21.3.1 Lipid-based drug delivery systems 482 Conflicts of interest 541
21.3.2 Protein-based drug delivery systems 484 References 542
21.3.3 Carbohydrate-based drug delivery Further reading 546
systems 486
21.4 Conclusions 488 24. Carbohydrates mimetics: enzyme
References 489 inhibitors and target molecules in several
diseases
22. Biological macromolecules in cell VERÓNICA E. MANZANO, CUSTODIANA A.
encapsulation COLMENAREZ LOBO AND EVANGELINA REPETTO
MILAN MILIVOJEVIC, IVANA PAJIC-LIJAKOVIC AND
BRANKO BUGARSKI 24.1 Introduction 547
24.1.1 Biomass and biobased materials 547
22.1 Introduction 491 24.1.2 Carbohydrates 548
22.2 Biopolymers used for cell encapsulation in 24.1.3 Biological and medicinal interest of
TE 496 carbohydrates 550
22.2.1 Agarose 496 24.1.4 Glycosidases 551
22.2.2 Alginate 497 24.2 Glycomimetics 554
22.2.3 Chitin and chitosan 499 24.2.1 Iminosugars 554
 Contents xi
24.2.2 Carbasugars 561 25.5.2 Gene delivery 592
24.2.3 Thiosugars 564 25.6 Macromolecules on tissue engineering 593
24.3 Hybrid carbohydrates 566 25.6.1 Wound management 597
24.4 Macromolecules 567 25.6.2 Development of skin substitutes 597
24.4.1 Multivalents 567 25.7 Conclusion 600
24.4.2 Polysaccharides 569 References 600
24.5 Conclusions 570
References 570 26. Future perspectives of biological
macromolecules in biomedicine
IV ANA R. NEVES, RÚBEN FARIA, TÂNIA ALBUQUERQUE,
TELMA QUINTELA, ÂNGELA SOUSA AND DIANA COSTA

Others 26.1 Bio-nanotechnology 607

25. Current challenging issues of
biological macromolecules in biomedicine
Y. DE ANDA-FLORES, E. CARVAJAL-MILLAN,
A.C. CAMPA-MADA, K.G. MARTÍNEZ-ROBINSON,
J. LIZARDI-MENDOZA, A. RASCÓN-CHU,
A.L. MARTÍNEZ-LÓPEZ AND J. TANORI-CORDOVA
25.1 Introduction 581
25.2 Biological macromolecules 582
25.3 Macromolecules in biomedical applications 583
25.4 Macromolecules in targeted drug delivery 584
25.5 Biomaterials as targeted drug delivery 585
25.5.1 Hydrogels for drug delivery 585
26.1.1 Delivery systems 608
26.2 Mitochondrial gene therapy 610
26.2.1 Mitochondrion 611
26.2.2 Mitochondrial mutations 611
26.2.3 Targeting Mitochondria 612
26.3 Crosstalk between chronobiology and cancer 616
26.3.1 Circadian clock and cancer
development 617
26.3.2 Chronobiology and cancer treatment 619
26.4 Concluding remarks 624
References 625
Index 633
 List of contributors
Ahmed S. Abo Dena Nanomedicine Laboratory,
Center for Materials Science, Zewail City of
Science and Technology, Giza, Egypt;
Department of Pharmaceutical Chemistry,
National Organization for Drug Control and
Research (NODCAR), Giza, Egypt
Caleb Acquah School of Nutrition Sciences,
Faculty of Health Sciences, University of Ottawa,
Ottawa, ON, Canada
Tanvir Ahmed Department of Applied Chemistry
and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh
Azilah Ajit Faculty of Chemical & Natural
Resources Engineering, Universiti Malaysia
Pahang, Kuantan, Malaysia
Yahya S. Al-Awthan Department of Biology,
Faculty of Science, University of Tabuk, Tabuk,
Saudia Arabia; Department of Biology, Faculty of
Science, Ibb University, Ibb, Yemen
Tânia Albuquerque CICS-UBI—Health Sciences
Research Centre, University of Beira Interior,
Covilhã, Portugal
Sukumaran Anil Department of Dentistry, Oral
Health Institute, Hamad Medical Corporation,
College of Dental Medicine, Qatar University,
Doha, Qatar
Nuvia Marina Apolonio–Hernandez Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
Leonard Ionut Atanase Academy of Romanian
Scientists, Bucuresti, Romania; Faculty of Dental
Medicine, “Apollonia” University of Iasi, Iasi,
Romania
Omar Bahattab Department of Biology, Faculty of
Science, University of Tabuk, Tabuk, Saudia Arabia
xiii
Anindita Behera School of Pharmaceutical
Sciences, Siksha “O” Anusandhan, Deemed to be
University, Bhubaneswar, India
Muhammad Bilal School of Life Science and Food
Engineering, Huaiyin Institute of Technology,
Huai’an, China
Branko Bugarski Department of Chemical
Engineering, Faculty of Technology and Metallurgy,
University of Belgrade, Belgrade, Serbia
A.C. Campa-Mada Biopolymers-CTAOA,
Research Center for Food and Development
(CIAD, A.C.), Hermosillo, Mexico
Ireri Alejandra Carbajal-Valenzuela Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
E. Carvajal-Millan Biopolymers-CTAOA, Research
Center for Food and Development (CIAD, A.C.),
Hermosillo, Mexico
Long Chen School of Food Science and
Technology, Jiangnan University, Wuxi, P.R.
China; State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi, P.R. China
Wai Teng Cheng School of Science, Monash
University Malaysia, Bandar Sunway, Malaysia
Custodiana A. Colmenarez Lobo Research Center
in Carbohydrate Chemistry (CIHIDECAR),
National Scientific and Technical Research
Council (CONICET)-UBA, Buenos Aires,
Argentina
Diana Costa CICS-UBI—Health Sciences Research
Centre, University of Beira Interior, Covilhã,
Portugal
Eduardo Costa Universidade Católica Portuguesa,
CBQF—Centro de Biotecnologia e Quı́mica Fina
—Laboratório Associado, Escola Superior de
Biotecnologia, Porto, Portugal
xiv List of contributors
Pandurang Appana Dalavi Biomaterials Research
Laboratory, Yenepoya Research Centre,
Yenepoya (Deemed to be University),
Deralakatte, Mangalore, India
Michael K. Danquah Chemical Engineering
Department, University of Tennessee,
Chattanooga, TN, United States
Y. De Anda-Flores Biopolymers-CTAOA, Research
Center for Food and Development (CIAD, A.C.),
Hermosillo, Mexico
Harshal Deshmukh Amity Institute of
Biotechnology, Amity University Maharashtra,
Mumbai, —Pune Expressway, Bhatan Post—
Somathne, Panvel, Mumbai, India
Amal Kumar Dhara Department of Pharmacy,
Contai Polytechnic, Contai, India
Ibrahim M. El-Sherbiny Nanomedicine
Laboratory, Center for Materials Science, Zewail
City of Science and Technology, Giza, Egypt
Rúben Faria CICS-UBI—Health Sciences Research
Centre, University of Beira Interior, Covilhã,
Portugal
Ana Angelica Feregrino-Pérez Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
M.D. Figueroa-Pizano Biopolymers-CTAOA,
Research Center for Food and Development
(CIAD), Hermosillo, Sonora, Mexico
D.A.S. Gamage Department of Chemical and
Process Engineering, Faculty of Engineering,
University of Peradeniya, Peradeniya, Sri
Lanka
Beatriz González-Arias Biosystems Engineering
Group, School of Engineering-Campus Amazcala,
Autonomous University of Querétaro (México),
Querétaro, México
Natanamurugaraj Govindan Algae Culture
Collection Center & Laboratory, Faculty of
Industrial Sciences and Technology, Universiti
Malaysia Pahang, Kuantan, Malaysia; Centre for
Research in Advanced Tropical Bioscience,
Universiti Malaysia Pahang, Kuantan, Malaysia
Ramón Gerardo Guevara-González Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
Diana Vanesa Gutierrez-Chavez Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
Md Saquib Hasnain Department of Pharmacy,
Palamau Institute of Pharmacy, Daltonganj, India
Shafiul Hossain Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh; Department of
Chemical Engineering and Polymer Science,
Shahjalal University of Science and Technology,
Sylhet, Bangladesh
Muhammad Imran University Institute of Diet &
Nutritional Sciences, Faculty of Allied Health
Sciences, The University of Lahore, Lahore,
Pakistan
Hafiz M.N. Iqbal Tecnologico de Monterrey,
School of Engineering and Sciences, Monterrey,
Mexico
Jaison Jeevanandam CQM—Centro de Quı́mica
da Madeira (Madeira Chemistry Center), MMRG,
Universidade da Madeira (University of
Madeira), Campus da Penteada (Penteada cam-
pus), Funchal, Portugal
Alejandra Jimenez-Hernandez Biosystems
Engineering Group, School of Engineering-
Campus Amazcala, Autonomous University of
Querétaro (México), Querétaro, México
Zhengyu Jin School of Food Science and
Technology, Jiangnan University, Wuxi, P.R.
China; State Key Laboratory of Food Science and
Technology, Jiangnan University, Wuxi, P.R.
China
Sumaya F. Kabir Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh
Krishnan Kanny Composite Research Group,
Department of Mechanical Engineering, Durban
University of Technology, Durban, South Africa
Jaya Lakkakula Amity Institute of Biotechnology,
Amity University Maharashtra, Mumbai, —Pune
Expressway, Bhatan Post—Somathne, Panvel,
Mumbai, India
J. Lizardi-Mendoza Biopolymers-CTAOA,
Research Center for Food and Development
(CIAD, A.C.), Hermosillo, Mexico
 List of contributors xv
Manuela Machado Universidade Católica Mohammad S. Mubarak Department of
Portuguesa, CBQF—Centro de Biotecnologia e Chemistry, The University of Jordan, Amman,
Quı́mica Fina—Laboratório Associado, Escola Jordan
Superior de Biotecnologia, Porto, Portugal Naveed Muhammad Department of Pharmacy,
T. Madhujith Department of Food Science and Abdul Wali Khan University, Khyber
Technology, Faculty of Agriculture, University of Pakhtunkhwa, Pakistan
Peradeniya, Peradeniya, Sri Lanka Hira Munir Department of Biochemistry and
Gaanty Pragas Maniam Algae Culture Collection Biotechnology, University of Gujrat, Gujrat,
Center & Laboratory, Faculty of Industrial Pakistan
Sciences and Technology, Universiti Malaysia Sesha Subramanian Murugan Biomaterials
Pahang, Kuantan, Malaysia; Centre for Research Research Laboratory, Yenepoya Research Centre,
in Advanced Tropical Bioscience, Universiti Yenepoya (Deemed to be University),
Malaysia Pahang, Kuantan, Malaysia Deralakatte, Mangalore, India
Verónica E. Manzano Faculty of Exact and Amit Kumar Nayak Department of
Natural Sciences, Department of Organic Pharmaceutics, Seemanta Institute of
Chemistry, University of Buenos Aires, Buenos Pharmaceutical Sciences, Jharpokharia, India
Aires, Argentina; Research Center in
Carbohydrate Chemistry (CIHIDECAR), National Ana R. Neves CICS-UBI—Health Sciences
Scientific and Technical Research Council Research Centre, University of Beira Interior,
(CONICET)-UBA, Buenos Aires, Argentina Covilhã, Portugal
Ahmed Olatunde Department of Biochemistry,
A.L. Martı́nez-López NANO-VAC Research
Abubakar Tafawa Balewa University, Bauchi,
Group, Department of Chemistry and
Nigeria
Pharmaceutical Technology, University of
Navarra, Pamplona, Spain Ivana Pajic-Lijakovic Department of Chemical
Engineering, Faculty of Technology and
K.G. Martı́nez-Robinson Biopolymers-CTAOA,
Metallurgy, University of Belgrade, Belgrade,
Research Center for Food and Development
Serbia
(CIAD, A.C.), Hermosillo, Mexico
Dilipkumar Pal Department of Pharmaceutical
David Julian McClements Department of Food Sciences, Guru Ghasidas Vishwavidyalaya (A
Science, University of Massachusetts, Amherst, Central University), Bilaspur, India
MA, United States
Aristeidis Papagiannopoulos Theoretical and
Ming Miao School of Food Science and Physical Chemistry Institute, National Hellenic
Technology, Jiangnan University, Wuxi, P.R. Research Foundation, Athens, Greece
China; State Key Laboratory of Food Science and
Manuela Pintado Universidade Católica
Technology, Jiangnan University, Wuxi, P.R.
Portuguesa, CBQF—Centro de Biotecnologia e
China
Quı́mica Fina—Laboratório Associado, Escola
Milan Milivojevic Department of Chemical Superior de Biotecnologia, Porto, Portugal
Engineering, Faculty of Technology and
Marcel Popa “Apollonia” University of Iasi,
Metallurgy, University of Belgrade, Belgrade,
Faculty of Dental Medicine, Iasi, Romania;
Serbia
Academy of Romanian Scientists, Bucuresti,
Md. Minhajul Islam Department of Applied Romania; Faculty of Chemical Engineering and
Chemistry and Chemical Engineering, Faculty of Environmental Protection, Department of Natural
Engineering and Technology, University of and Synthetic Polymers, “Gheorghe Asachi”
Dhaka, Dhaka, Bangladesh Technical University of Iasi, Iasi, Romania
Aurelie Sarah Mok Tsze Chung School of Science, Sarmad Ahmad Qamar Department of
Monash University Malaysia, Bandar Sunway, Biochemistry, University of Agriculture,
Malaysia Faisalabad, Pakistan
xvi List of contributors
Telma Quintela CICS-UBI—Health Sciences
Research Centre, University of Beira Interior,
Covilhã, Portugal
Stefania Racovita “Petru Poni” Institute of
Macromolecular Chemistry, Iasi, Romania
Hamza Rafeeq Department of Biochemistry,
University of Agriculture, Faisalabad, Pakistan
Mohd Hasbi Ab. Rahim Algae Culture Collection
Center & Laboratory, Faculty of Industrial
Sciences and Technology, Universiti Malaysia
Pahang, Kuantan, Malaysia; Centre for Research
in Advanced Tropical Bioscience, Universiti
Malaysia Pahang, Kuantan, Malaysia
Ashiqur Rahman Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh; National Institute of
Textile Engineering and Research (NITER),
Dhaka, Bangladesh
Mohammed Mizanur Rahman Department of
Applied Chemistry and Chemical Engineering,
Faculty of Engineering and Technology,
University of Dhaka, Dhaka, Bangladesh
Khushboo Raj School of Pharmacy, Arka Jain uni-
versity, Tata, Jamshedpur, India
Ajay Vasudeo Rane Composite Research Group,
Department of Mechanical Engineering, Durban
University of Technology, Durban, South Africa
A. Rascón-Chu Biotechnology-CTAOV, Research
Center for Food and Development (CIAD, A.C.),
Hermosillo, Mexico
Abdur Rauf Department of Chemistry, University
of Swabi Anbar, Khyber Pakhtunkhwa, Pakistan
Evangelina Repetto Faculty of Exact and Natural
Sciences, Department of Organic Chemistry,
University of Buenos Aires, Buenos Aires,
Argentina; Research Center in Carbohydrate
Chemistry (CIHIDECAR), National Scientific and
Technical Research Council (CONICET)-UBA,
Buenos Aires, Argentina
Enrique Rico-Garcı́a Biosystems Engineering
Group, School of Engineering-Campus Amazcala,
Autonomous University of Querétaro (México),
Querétaro, México
Supriyo Saha School of Pharmaceutical Sciences
and Technology, Sardar Bhagwan Singh
University, Dehradun, India
Md. Sazedul Islam Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh
Md. Shahruzzaman Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh
Sara Silva Universidade Católica Portuguesa,
CBQF—Centro de Biotecnologia e Quı́mica Fina
—Laboratório Associado, Escola Superior de
Biotecnologia, Porto, Portugal
Ângela Sousa CICS-UBI—Health Sciences
Research Centre, University of Beira Interior,
Covilhã, Portugal
Ahmad Ziad Sulaiman Faculty of Bio-Engineering
& Technology, University Malaysia Kelantan
Kampus Jeli, Kelantan, Malaysia
Sabrina Sultana Department of Applied
Chemistry and Chemical Engineering, Faculty of
Engineering and Technology, University of
Dhaka, Dhaka, Bangladesh; Department of Arts
and Sciences, Ahsanullah University of Science
and Technology, Dhaka, Bangladesh
Joash Ban Lee Tan School of Science, Monash
University Malaysia, Bandar Sunway, Malaysia
J. Tanori-Cordova Department of Polymers and
Materials Research, University of Sonora,
Hermosillo, Mexico
Yong Kiat Teo School of Science, Monash
University Malaysia, Bandar Sunway, Malaysia
Himja Tiwari Amity Institute of Biotechnology,
Amity University Maharashtra, Mumbai, —Pune
Expressway, Bhatan Post—Somathne, Panvel,
Mumbai, India
Irineo torres-Pacheco Biosystems Engineering
Group, School of Engineering-Campus Amazcala,
Autonomous University of Querétaro (México),
Querétaro, México
Tabussam Tufail University Institute of Diet &
Nutritional Sciences, Faculty of Allied Health
Sciences, The University of Lahore, Lahore,
Pakistan
 List of contributors xvii
Silvia Vasiliu “Petru Poni” Institute of N.E. Wedamulla Department of Export
Macromolecular Chemistry, Iasi, Romania Agriculture, Faculty of Animal Science and
Jayachandran Venkatesan Biomaterials Research Export Agriculture, Uva Wellassa University,
Laboratory, Yenepoya Research Centre, Badulla, Sri Lanka
Yenepoya (Deemed to be University), Deepti Yadav Department of Biotechnology and
Deralakatte, Mangalore, India Food Science, Durban University of Technology,
Eleni Vlassi Theoretical and Physical Chemistry Durban, South Africa
Institute, National Hellenic Research Foundation, Zhongyu Yang School of Food Science and
Athens, Greece Technology, Jiangnan University, Wuxi, P.R.
Nilesh Shirish Wagh Amity Institute of China
Biotechnology, Amity University Maharashtra,
Mumbai, —Pune Expressway, Bhatan Post—
Somathne, Panvel, Mumbai, India
 Preface
The scope of this book, entitled Biological
Macromolecules: Bioactivity and Biomedical
Applications, is the coverage and review of
recent trends and applications of biological
macromolecules, such as carbohydrates, lipids,
proteins, peptides, and nucleic acids in biome-
dicines, drug delivery, growth factors delivery,
nutrients and nucleic acids delivery, cell encap-
sulation, enzyme mobilization, and tissue
engineering.
The mysteries of life lie in biological macro-
molecules. A large volume of biological macro-
molecules is obtained from different biological
origins such as plants, algae, fungi, animals,
and microbial sources. Biological macromole-
cules exhibit some significant and favorable
advantages over synthetic macromolecules,
such as sustainable and economic production,
biocompatibility, biodegradability, and
improved bioavailability. In recent years, a
plethora of biological macromolecules (carbo-
hydrates, lipids, proteins, peptides, and nucleic
acids) has been used in the biomedical and
healthcare fields. They showed varieties of
bioactivities such as antioxidant, anticancer,
antidiabetic, antimicrobial, immunomodula-
tory activities on the central nervous system,
and gastrointestinal activity. The other biomed-
ical applications include drug delivery, growth
factors delivery, nutrients and nucleic acids
delivery, cell encapsulation, enzyme mobiliza-
tion, and tissue engineering. The structure-
property relationship is also an important
aspect for a thorough understanding of the bio-
activity of biological macromolecules.
xix
This book, containing 4 sections and 26
chapters, provides a systematic insight into the
inclusive discussions on bioactivity and bio-
medical applications of different biological
macromolecules. We are glad to see that many
authors across the globe accepted our invita-
tion and contributed valued chapters for this
book, covering a wide spectrum of fields. A
concise account of the contents of each chapter
has been described to provide a glimpse of the
book to the potential readers of various fields.
The topics in the book (in order of prefer-
ence) include the following: Biological
Macromolecules: Sources, Properties, and functions
(Chapter 1)—this chapter describes sources
physicochemical properties, bioactivity and
biomedical applications of different biological
macromolecules concisely; Structure Activity
Relationship of Biological Macromolecules
(Chapter 2)—this chapter aims to provide an
overview of the structural features influencing
the bioactivities of biological macromolecules,
namely L-amino acid oxidases, lysostaphin,
and metallo-β-lactamase-such as lactonase and
chitosan; The Importance of Biological
Macromolecules in Biomedicine (Chapter 3)—this
chapter highlights the therapeutic aspects of
macromolecules and the medicinal use of bio-
logical macromolecules against various dis-
eases and ailments; Modification Techniques for
Carbohydrate Macromolecules (Chapter 4)—this
chapter characteristically abridges the signifi-
cant developments of the last five to ten years
and discusses critically in the area of modifica-
tion of carbohydrates macromolecules;
xx Preface
Biological Macromolecules as Nutraceuticals
(Chapter 5)—this chapter aims to demonstrate
some recent knowledge regarding the nutra-
ceutical and biological activities of the macro-
molecules of biological origin, as well as some
frontier applications of these in healthcare;
Biological Macromolecules as Antioxidants
(Chapter 6)—this chapter highlights the poten-
tial applications of biological macromolecules
as antioxidants to scavenge reactive oxygen
species and control oxidative stress, which
leads to various pathogenesis; Biological
Macromolecules as Antimicrobial Agents
(Chapter 7)—the chapter describes the antimi-
crobial activity of biological macromolecules
(chitosan, cellulose, alginate, gelatin, collagen,
and keratin) and also, comprehensively eluci-
dates their applications in addressing chal-
lenges associated with drug delivery, wound
dressing, food packaging, and so on Biological
Macromolecules From Algae and Their
Antimicrobial Applications (Chapter 8) this
chapter provides an overview of bioactive
macromolecules and their antimicrobial activi-
ties with particular reference to algal sources;
Biological Macromolecules Acting on Central
Nervous System (Chapter 9)—in this chapter,
the role of biological macromolecules on cen-
tral nervous system and their critical role in
downregulation after the various neurological
disorders have been discussed; Biological
Macromolecules as Antidiabetic Agents
(Chapter 10)—this chapter is an overview of
different types of biological macromolecules
and their applications as potential antidiabetic
agents and also, highlights the advantages, lim-
itations and future perspectives of biological
macromolecules as antidiabetic agents;
Biological Macromolecules as Anticancer Agents
(Chapter 11)—this chapter presents the extrac-
tion of macromolecules such as carbohydrate,
proteins, lipids, and nucleic acid (miRNAs)
from different biological sources, such as
plants, animal, algae and fungi. The various
mechanisms by which the macromolecules
exhibit their anticancer activity have been
discussed briefly along with several assays
done to evaluate cytotoxicity of the macromole-
cules against various cancers such as lung can-
cer, breast cancer, cervical cancer, and colon
cancer. Biological Macromolecules as
Immunomodulators (Chapter 12)—this topic
focuses on the potential modulations of
immune response of biomacromolecules (three
major classes of compounds: lipids, proteins
and polysaccharides); Biological Macromolecules
Acting on Gastrointestinal Systems
(Chapter 13)—this chapter describes the role of
biological macromolecules for the management
of gastrointestinal system and related disor-
ders; Synthetic Macromolecules With Biological
Activity (Chapter 14)—this chapter describes
some classes of synthetic macromolecules with
biological activity that have a great importance
on the human comfort and health, including
antimicrobial polymers, antioxidant polymers,
and polymeric sequestrants; Biological
Macromolecules in Drug Delivery (Chapter 15)—
this chapter focuses on the advancements in
the uses of various biological macromolecules
in drug delivery applications; Biological macro-
molecules in tissue engineering (Chapter 16)—this
chapter provides an overview on the important
role of natural-derived biomaterials (alginate,
chitosan, carrageenan, fucoidan, ulvan, colla-
gen, and gelatin) combining with ceramic bio-
materials for bone tissue construction;
Biological Macromolecules for Drug Delivery in
Tissue Engineering (Chapter 17)—This chapter
is focused on the preparation and physico-
chemical characterization of engineered bioma-
terials, based on biological macromolecules
(polysaccharides and proteins), as scaffolds
which are capable of supporting physiological
activities of cells, but also can act as drug deliv-
ery systems for tissue engineering and wound
healing; Biological Macromolecules for Growth
Factor Delivery (Chapter 18)—this chapter dis-
cusses the fabrication of synthetic and natural
macromolecules, sometimes combined with
other mineral or metallic compounds for
growth factor delivery; Biological
 Preface
Macromolecules for Growth Factor Delivery in
Bone Regeneration (Chapter 19)—this chapter
describes the process of bone tissue regenera-
tion in healing injuries and arthritic conditions,
introduces the main ideas through the scope of
allogenous and autogenous transplantation
and demonstrates the role of growth factors in
these processes; Biological Macromolecules for
Nutrients Delivery (Chapter 20)—This chapter
focuses on the types of nutrients that need to
be delivered, the biological macromolecules
that can be used to construct edible delivery
systems, the most common delivery systems
currently used for this purpose, and some of
the major challenges that must be addressed in
the future; Biological Macromolecules for Nucleic
Acid Delivery (Chapter 21)—this chapter
describes the nonviral nucleic acid delivery
systems made up of biological macromole-
cules, such as peptides, lipids, and carbohy-
drates and also gives an introduction on the
history and structure of nucleic acids; Biological
Macromolecules in Cell Encapsulation
(Chapter 22)—this chapter aims to review the
most examined, most promising and recently
proposed biopolymers that are used in tissue
engineering scaffolds, and to highlight their
main properties, drawbacks, fields of applica-
tions and fabrication technologies in order to
provide readers with important guidelines for
selecting appropriate scaffold biomaterials;
Biological Macromolecules for Enzyme
Immobilization (Chapter 23)—this chapter pro-
vides a broad overview of properties and the
applications of various naturally occurring bio-
polymers, that is, chitosan, chitin, agarose, algi-
nates, cellulose, gelatin, dextran, carrageenan,
pectin and xanthan gum for their applications
in enzyme immobilization with recent litera-
ture studies indicating biopolymer-based sup-
port material development and their utilization
to make biocatalysts with desired stability and
catalytic functionalities; Carbohydrates for
Enzyme Inhibition and Their Use as Target
Molecules for the Interference of Diseases
(Chapter 24)—this chapter describes the study
of a widespread group of enzymes and the
xxi
inhibition of these enzymes constitutes an
interesting and novel strategy to approach new
therapies against numerous diseases; Current
Challenging Issues of Biological Macromolecules in
Biomedicine (Chapter 25)—this chapter provides
information on recent innovations in various
biomaterials, engineered from macromolecules
ranging from drug delivery, cancer therapies,
tissue engineering, bioprinting and wound
healing; Future Perspectives of Biological
Macromolecules in Biomedicine (Chapter 26)—
this chapter discusses the impact of the combi-
nation of nanotechnology and chronobiology
in personalized cancer treatment.
We sincerely acknowledge the valuable con-
tribution of the distinguished authors and con-
vey our sincere thanks. This book could not
have been published without the cooperation
of Barbara Makinster, Editorial Project
Manager. We wish to express our cordial grati-
tude to Elsevier Inc., Michelle Fisher
(Acquisition Editor), and other editorial staff
for their invaluable supports in organizing the
intelligent editing of the book. We also grate-
fully acknowledge all the permissions we
received for reproducing the copyright materi-
als from different sources. Finally, we cannot
overlook the sacrifices and supports from our
family members during the preparation of the
current book. All our friends, colleagues, and
students who have helped in the process of
editing this book deserve our great apprecia-
tion. Contributing authors, the publishers, and
we, the editors, will be extremely happy if our
endeavor fulfills the needs of the academicians,
researchers, students, pharmaceutical experts,
biomedicine experts, and formulators.
Amit Kumar Nayak1, Amal Kumar Dhara2
and Dilipkumar Pal3
1
Department of Pharmaceutics, Seemanta Institute
of Pharmaceutical Sciences, Jharpokharia, India
2
Department of Pharmacy, Contai Polytechnic,
Govt. of West Bengal, Contai, India 3Department of
Pharmaceutical Sciences, Guru Ghasidas
Vishwavidyalaya (A Central University), Bilaspur,
India
 C H A P T E R
1
Biological macromolecules: sources,
properties, and functions
Amal Kumar Dhara1 and Amit Kumar Nayak2
1
Department of Pharmacy, Contai Polytechnic, Contai, India 2Department of Pharmaceutics, Seemanta
Institute of Pharmaceutical Sciences, Jharpokharia, India
1.1 Introduction
The mystery of life is in biological macromo-
lecules. There are four important classes of bio-
logical macromolecules, viz., carbohydrates,
lipids, proteins, and nucleic acids (Luo, Zhang,
Wu, Liang, & Li, 2020; Zhang, Sun, & Jiang,
2018). Carbohydrates, proteins, and nucleic
acids naturally exist as long chain polymers,
while lipids are smaller and in true sense, these
are all considered as biopolymers (Albertsson,
2019; Teramoto, 2020; Zhang et al., 2018).
Carbohydrates are the storage form of energy
and meet the demand as and when required
(Slavin & Carlson, 2014). Lipids are also stor-
age form of energy and are the important
structural components of the cell membrane
(van Meer, Voelker, & Feigenson, 2008; Zheng,
Fleith, Giuffrida, O’Neill, & Schneider, 2019).
Proteins serve several functions including
structural support, catalyzing important meta-
bolic reactions, signals receiving and transmis-
sion, etc. (Watford & Wu, 2018; Zaretsky &
Wreschner, 2008). Nucleic acids, that is, deox-
yribonucleic acid (DNA) and ribonucleic acid
Biological Macromolecules
DOI: https://doi.org/10.1016/B978-0-323-85759-8.00005-1
(RNA) are responsible for carrying genetic
blueprint and information for protein synthesis
(Minchin & Lodge, 2019; Schwartz, Schwartz,
Mieszerski, McNally, & Kobilinsky, 1991).
Biological macromolecules are abundantly
available in nature and also possess properties
like biocompatibility, environmental friendly,
biodegradability, etc., because of their natural
sources (Chandika et al., 2020; Teramoto, 2020).
Various species of algae have been mentioned
to be used as bioactive compounds and are
also employed as antibacterial agents (Shannon
& Abu-Ghannam, 2016). Various disorders
related to central nervous system, such as
Alzheimer’s disease, Parkinson’s disease, con-
vulsive disorders, etc., are being treated with
the biological macromolecules (Acosta &
Cramer, 2020; Soderquist & Mahoney, 2010;
Zhang et al., 2018). All the biological macromo-
lecules, viz., carbohydrates, lipids, proteins
and nucleic acids, have shown their significant
role in the management of cancer therapy and
have been advocated to be used against vari-
ous cancers like lung cancer, colon cancer,
breast cancer, cervical cancer, etc.
3 © 2022 Elsevier Inc. All rights reserved.
4 1. Biological macromolecules: sources, properties, and functions
(Corn, Windham, & Rafat, 2020; Oana,
Adriana, Mircea, Dragos, & Monica, 2018;
Rodrigues Mantuano, Natoli, Zippelius, &
Läubli, 2020; Sun, Jing, Ma, Feng, & Hu, 2020).
Another important area of research is immuno-
modulators with respect to present SARS-CoV-2
perspective, where the biological macromole-
cules, mainly proteins, play significant role (Ji
et al., 2020). Proteins are associated with the
development process of immune system (Daly,
Reynolds, Sigal, Shou, & Liberman, 1990).
Lipids are also responsible to play key role as
adjuvants for the development of vaccines
(Martinez-Gil, Goff, & Tan, 2018; Schwendener,
2014). Day by day, the incidence of lifestyle
diseases more specifically diabetes, hyperten-
sion, etc., are increasing vertically, where the
roles of biological macromolecules have been
studied and were found to be utilized widely
as antidiabetic agents (Alam, Shafique, Amjad,
& Bin Asad, 2019; Hu, Nie, & Xie, 2018; Rı́os,
Francini, & Schinella, 2015; Yu, Shen, Song, &
Xie, 2018). They cause increase in insulin secre-
tion and thus, reduce the blood glucose level
(Rı́os et al., 2015). Chitosan is a well-known
polysaccharide, which is reported to exhibit
antimicrobial and antidiabetic activities
(Hasnain & Nayak, 2018; Karadeniz & Kim,
2014; Rabea, Badawy, Stevens, Smagghe, &
Steurbaut, 2003). Extensive research is going
on in the area of tissue engineering for the
development of artificial tissue to repair and
replace defective or diseased tissue or organs
(Hasnain, Ahmad, Chaudhary, Hoda, &
Nayak, 2019; Nayak, Ahmed, Tabish, &
Hasnain, 2019; Pal, Saha, Nayak, & Hasnain,
2019). Naturally derived biological macromole-
cules like chitosan, alginate, carrageenan,
ulvan, gelatin, etc., have been used for
bone tissue regeneration (Hasnain, Nayak,
Singh, & Ahmad, 2010; Maity, Hasnain, Nayak,
& Aminabavi, 2021; Nayak, Ahmed, Tabish, &
Hasnain, 2019). Numbers of polysaccharide
have long been used in different types of drug
delivery systems as biopolymeric excipients
I. Background
(Hasnain et al., 2020; Kandar, Hasnain, &
Nayak, 2021; Maity et al., 2021; Nayak &
Hasnain, 2019a, 2019b; Nayak, Hasnain, Dhara,
& Pal, 2021; Pal, Saha, Nayak et al., 2019).
Polysaccharide and proteins are used exten-
sively for the preparation of hydrogels for
drug delivery, tissue regeneration, wound
dressings, etc. (Del Valle, Dı́az, & Puiggalı́,
2017; Nayak & Pal, 2016b; Nayak, Hasnain,
Pal, Banerjee, & Pal, 2020; Pal, Nayak, & Saha,
2019a, 2019b; Ray et al., 2020). Beside drug
delivery, the biological macromolecules have
continuously been used to formulate delivery
carrier-systems for growth factors (Rao, Rekha,
Anil, Lowe, & Venkatesan, 2019; Shariatinia,
2019). Polysaccharides are also employed for
the encapsulation of various bioactive sub-
stances like vitamins and nutraceuticals (Bala,
Singha, & Patra, 2019; Lauro, Amato, Sansone,
Carbone, & Puglisi, 2019). The current chapter
deals with a brief discussion about the sources,
properties, and valuable applications of vari-
ous biological macromolecules like carbohy-
drates, lipids, proteins and nucleic acids.
1.2 Carbohydrates
The most widely found organic compounds
in nature are carbohydrates. These are well-
known as very essential source of life or sus-
taining life itself (Slavin & Carlson, 2014).
Carbohydrates are commonly found in plants,
microorganisms and animal tissues (Werz &
Seeberger, 2005). These are also present in
blood, tissue fluids, etc. (Kilcoyne & Joshi,
2007). Carbon, hydrogen and oxygen are the
three primary elements of molecular structure
of carbohydrates (Luo et al., 2020; Werz &
Seeberger, 2005). These are optically active
polyhydroxy aldehydes or ketones. There are
three major classes of carbohydrates, broadly,
monosaccharides, oligosaccharides and poly-
saccharides (Slavin & Carlson, 2014).
 1.2 Carbohydrates
1.2.1 Monosaccharides
These are generally called simple sugars,
and the most common monosaccharide is glu-
cose. Most of the monosaccharides comprise of
the general formula CnH2nOn (Pigman &
Horton, 1972). The different classes of mono-
saccharides include aldoses (functional group
is aldehyde), and ketoses (functional group is
keto) (Slavin & Carlson, 2014; Werz &
Seeberger, 2005). On the basis of number of
carbon in the sugar, they are also subcategor-
ized into (Shin & Kim, 2013; Slavin & Carlson,
2014):
1. Trioses (containing three carbon atoms in
the sugar), for example, glyceraldehydes
and dihydroacetone,
2. Tetroses (containing four carbon atoms in
the sugar), for example, erythrose and
erythrulose,
3. Pentoses (containing five carbon atoms in
the sugar), for example, ribose and xylulose,
4. Hexoses (containing six carbon atoms in the
sugar), for example, glucose, glactose,
fructose and mannose, and
5. Heptoses (containing seven carbon atoms in
the sugar), for example, sedoheptulose.
Physicochemical properties of monosacchar-
ides: These are soluble in water, sweet in taste
and permeable through plasma membrane.
Monosaccharides react with hydrazine to form
osazones (Pigman & Horton, 1972). They
undergo reduction and form sugar alcohols
(e.g., glucose-sorbitol; fructose-mannitol; galac-
tose-dulcitol; glyceraldehyde-glycerol, etc.). On
oxidation, these produce sugar acids like glu-
conic acid. Monosaccharides play varieties of
important physiological functions (Pigman &
Horton, 1972). These are used for energy pro-
duction in living organisms. The vital compo-
nents of cells are RNA and DNA, which are
composed of ribose and deoxyribose and are
well-recognized as the building blocks of life
(Minchin & Lodge, 2019).
I. Background
5
1.2.2 Oligosaccharides
These yield 2
10 monosachharides on
hydrolysis. On the basis of number of mono-
saccharide units present, these are further sub-
classified into (Shin & Kim, 2013; Slavin &
Carlson, 2014): disaccharides (e.g., sucrose,
maltose, lactose and trehalose), trisaccharides
(e.g., raffinose and maltotriose), etc. These can
exhibit reducing property, when these contain
free aldehyde and/or ketone group, which is/
are not participated in the formation of
linkage.
1.2.3 Polysaccharides
These are formed by uniting monosaccha-
ride or there derivatives. These are joined
together by glycosidic linkage (Maity et al.,
2021). Unlike proteins and nucleic acids, poly-
saccharides exist as both linear as well as
branched polymers. These are colloidal in
nature. Polysaccharides are grouped into two
categories (Shin & Kim, 2013; Slavin & Carlson,
2014):
1. Homopolysaccharides: These yield one type
of monosaccharides on hydrolysis, for
example, starch, cellulose, glycogen, etc.
2. Heteropolysaccharides: These yield two or
more different type of monosaccharides on
hydrolysis, for example, hyaluronic acid,
heparin, chondriotin sulfate, etc.
Some important homopolysaccharides are
described here:
1. Starches—These contain several units of
glucose joined in α-1, 4-linkages and are
well-known as examples of
homopolysaccharides (Nayak & Pal, 2017).
In plants, it is the storage form of
carbohydrates. Different parts of the plants
are rich in starches like tubers, roots,
vegetables, cereals, etc. (Nayak & Pal, 2017;
Nayak, Bera, & Hasnain, 2020). In higher
6 1. Biological macromolecules: sources, properties, and functions
animals, starches are the most important
source of food. These consist of two types of
molecules (Fig. 1.1): (A) linear and water
soluble component, e.g., amylose and (B)
branched water insoluble, e.g., amylopectin.
Commercially, starch is produced mostly
from corn, but wheat starch, potato starch,
and tapioca starch are also used (Nayak
et al., 2020).
2. Cellulose—It is the chief constituent of
fibrous parts of the plants and consequently,
is the most abundant organic material
occurring in nature (Pal et al., 2019b). It is
made up of long chains of β D-glucose
molecules linked by 1, 4-linkages (Fig. 1.2). It
serves as bulk forming agent of the food.
Undigested cellulose increases the bulk of
feces and helps in the evacuation of bowels.
Cellulose exhibits some of important
FIGURE 1.1 Molecular structure of starch: (A) linear and water soluble component: amylose and (B) branched water
insoluble: amylopectin.
I. Background
properties like low density, flexibility, high
strength, biocompatibility, biodegradability,
etc., which suggest for biomedical
applications (Hasnain et al., 2020; Kandar
et al., 2021). In traditional healthcare,
cellulosic biomaterials play an important role
and recently, some significant areas of
application are being explored with the uses
of cellulose like drug delivery (Hasnain et al.,
2020; Kandar et al., 2021; Pal et al., 2019b),
tissue engineering (Hasnain, Nayak, Singh, &
Ahmad, 2010; Murizan, Mustafa, Ngadiman,
Mohd Yusof, & Idris, 2020), management of
wound (Alven & Aderibigbe, 2020), etc. By
using quaternary ammonium salt the surface
of cellulose nanocrystals (CNC) is modified,
which can inhibit the growth of Staphylococcus
aureus and Escherichia coli (Tavakolian, Jafari,
& van de Ven, 2020).
 1.2 Carbohydrates
FIGURE 1.3 Molecular structure of dextran.
3. Dextran—It is a highly branched polymer of
glucose, produced by yeast or bacteria
(Chen, Huang, & Huang, 2020; Huang &
Huang, 2018). The linear chain dextran
molecular structure are formed by 1, 6-α
glycosidic linkages (Fig. 1.3). Dextran occurs
in honey, maple syrup, etc. It is used as
plasma substitutes as it retains water in
circulation for longer period, when
administered intravenously (i.v.). Dextran
and its derivatives are being used to
formulate nanocarriers for advanced drug
delivery applications (Huang & Huang,
2018).
I. Background
7
FIGURE 1.2 Molecular
structure of cellulose.
4. Pectins—These form gel with sugar
solutions. In nature, pectins are usually
found in pulps and peels of citrus fruits,
apple pomaces, beet roots, etc. (Nayak &
Pal, 2016a). Chemically pectins are
polysaccharide of galacturonic acid,
galactose, and arabinose (Hasnain et al.,
2020; Kandar et al., 2021). Molecular
structure of pectin is presented in Fig. 1.4.
Pectins have been found to possess
beneficial biological activities, like
antioxidant, antiinflammatory, antibacterial,
immune regulation, and anticoagulation
activities (Rascoń-Chu, Gomez-Rodriguez,
Carvajal-Millan, & Campa-Mada, 2019).
Biomedical applications of pectins include
tissue engineering, drug delivery, wound
healing and gene delivery (Hasnain et al.,
2020; Kandar et al., 2021; Maity et al., 2021;
Nayak et al., 2019, 2021; Rascoń-Chu et al.,
2019).
5. Gum acacia or Gum Arabic—It is a plant-
derived gum containing hexoses or pentoses
or both. It is extensively used in
pharmaceutical, food and cosmetic
industries (Nayak, Das, & Maji, 2012). It is
an effective and useful excipient for the
preparation of nanomaterials for drug
delivery (De, Nayak, Kundu, Das, &
Samanta, 2021).
6. Alginates—These consist of linear polymer
of β (1-4)-linked D-mannuronic acid
(M-unit) and α (1-4)-linked L-guluronic
8 1. Biological macromolecules: sources, properties, and functions
FIGURE 1.4 Molecular structure of pectin.
acid (G-unit) (Hasnain et al., 2020; Kandar
et al., 2021; Nayak et al., 2021). Molecular
structure of pectin is presented in Fig. 1.5. It
is processed from marine algae and giant
kelp as raw materials. These are widely
used as thickener, emulsifier, stabilizer, etc.
(Kandar et al., 2021). Alginate based drugs
are effectively used for antimicrobial as well
as antiviral therapy (Szekalska, Pucilowska,
Szymańska, Ciosek, & Winnicka, 2016). The
alginate film with EDTA exhibited stronger
antimicrobial effects against Gram-negative
bacteria, especially, in case of processed
food packaging (Senturk Parreidt, Müller, &
Schmid, 2018). Structural modifications of
alginates can easily be made by using
crosslinkers, improvise the mechanical
strength and cell affinity and was widely
I. Background
FIGURE 1.5 Molecular structure of alginate.
used in the biomedical applications mainly
in drug delivery and tissue regeneration
(Malakar, Nayak, Jana, & Pal, 2013; Malakar,
Nayak, & Das, 2013).
7. Chitosan—It is a cationic natured
carbohydrate polysaccharide, extracted by
deacetylation of chitin (Hasnain & Nayak,
2018). It is reported to show various
biological properties including antidiabetic,
antioxidant, immune-enhancing,
antimicrobial as well as anticancer activities
(Hasnain & Nayak, 2018; Rabea et al., 2003;
Rı́os et al., 2015). Chitosan is very much
effective in the formulation of insulin with
controlled delivery functionality at the target
site (Barbosa et al., 2020). Carboxymethyl-
hexanoyl derivative and polyethylene glycol-
trimethyl complexes of chitosan have been
 1.3 Lipids
found to possess fat-lowering and fat-
preventing properties. Chitosan-based
collagen complex sponges showed
effectiveness in the healing of diabetic
wounds (Wang et al., 2008). Chitosan and its
derivatives are being extremely used in many
biomedical applications, such as drug
delivery, tissue engineering, wound dressing,
orthopedics, etc. (Hasnain et al., 2020;
Hasnain, Ahmad, Chaudhary, Hoda, &
Nayak, 2019; Hasnain, Nayak, Singh, &
Ahmad, 2010; Kandar, Hasnain, & Nayak,
2021; Maity, Hasnain, Nayak, & Aminabavi,
2021; Nayak, Ahmed, Tabish, & Hasnain,
2019; Pal, Saha, Nayak, & Hasnain, 2019).
8. Agar—It is a natural polysaccharide
obtained from seaweeds. It is a sulfuric acid
ester of a complex galactose polysaccharide
(Kandar et al., 2021). It is nondigestible
material and is used as a bulk laxative.
Recent years, a number of biocompatible
agar-based composite has been formulated
for their potential applications in biomedical
fields including drug delivery and tissue
engineering applications (Kandar et al.,
2021; Nayak, Alkahtani, & Hasnain, 2021;
Shah et al., 2019).
TABLE 1.1 Sources and functions of various polysaccharides.
Polysaccharides Source type
Cellulose Plants
Starches Plants
Pectins Plants
Carrageenan Microorganism
Alginate Microorganism
Hyaluronan Animals
Heparin Animals
Chondroitin sulfate Animals
Chitin and chitosan Animals
I. Background
9
9. Glycogen—This is also known as animal
starch, as it is a principal polysaccharide
occurring in animal tissues, specifically in
liver and muscle (Roach, Depaoli-Roach,
Hurley, & Tagliabracci, 2012). Glycogen is
the storage form of energy release, quickly,
when needed (Kreitzman, Coxon, & Szaz,
1992). Similar to starch, this is also
composed of glucose units united by 1, 4-
linkages and branches arising by 1, 6-
linkages.
Sources and functions of various polysac-
charides are listed in Table 1.1.
1.3 Lipids
Lipids are heterogeneous group of organic
compounds, related either actually or poten-
tially, to the fatty acids (Pandey & Kohli, 2018).
They are poorly soluble in water and soluble in
nonpolar solvents like chloroform, benzene,
petroleum ether, etc. In our body, lipids are an
integral part of the cell membrane structure,
metabolic fuel and storage form of energy (van
Meer et al., 2008; Zheng et al., 2019). Lipids act
Functions
Cell structure and food additives
Storage and drug adjuvants
Food additives
Food additives
Drug adjuvant
Animal tissue structure, therapeutic agents
Animal tissue structure, therapeutic agents
Animal tissue structure
Tissue scaffolds
10 1. Biological macromolecules: sources, properties, and functions
as mechanical, thermal and electrical insulators
(Pandey & Kohli, 2018). Lipids are usually clas-
sified as follows:
1.3.1 Simple lipids
These are esters of fatty acids with certain
alcohols, generally glycerol. According to
nature of alcohols, these are (Vance & Vance,
2002):
1. Fats and oils: Ester of fatty acids with
glycerol. These are also known as natural fat
or triglyceride or triacylglycerol. At room
temperature when these are solid known as
fat and when liquid these are known as oil.
2. Waxes: These are esters of fatty acid with
higher molecular weight monohydric
alcohol.
1.3.2 Compound or conjugate lipids
These are ester of fatty acids containing
groups, in addition to an alcohol and the fatty
acids. These are further classified as (Vance &
Vance, 2002):
1. Phospholipids: These contain fatty acids,
glycerol, a phosphoric acid residue and
sometimes a nitrogenous base. They are
subdivided as:
a. Phosphotidic acids—on hydrolysis, these
produce one molecule each of glycerol
and phosphoric acid with two molecules
of fatty acids.
b. Lecithins—contain glycerol, fatty acid,
phosphoric acid and the nitrogen base
choline. These are widely distributed in
different animal tissues including brain,
liver, blood, cardiac muscles, etc., and also
found in plant seeds. Lecithins are used as
emulsifying and smoothing agents in food
industry (Robert, Couëdelo, Vaysse, &
Michalski, 2020). Lecithins have been
mentioned to be used for the
I. Background
development of nanocarriers for drug
delivery (Das, Sen, Maji, Nayak, & Sen,
2017; Malakar, Sen, Nayak, & Sen, 2012).
c. Cephalins—similar in structure with
lecithin but the base is ethanolamine.
These are found in brain, liver, cardiac
muscles, erythrocytes, etc.
d. Plasmalogens—found in brain, cardiac
muscles, erythrocytes, etc.
e. Sphingomyelins—on hydrolysis, these
give a single fatty acid, a nitrogen base
sphingosine, phosphoric acid and choline
but no glycerol. These participate in
different signaling pathways.
2. Glycolipids: These contain sphingol, a
carbohydrate (galactose), and fatty acid.
Large amount of glycolipids are present in
the white matter of brain and in the myelin
sheath of nerves (Willison, 2018).
3. Sulpholipids: Lipid material containing
sulfur has long been known to be present in
the different tissues like liver, kidneys,
brain, etc. It is found in the white matter of
the brain.
4. Lipoproteins: These are composed of
lipid material bound to the protein. The
lipid of lipoproteins mainly consists of
cholesterol esters and phospholipids (such
as stearic, palmitic, and oleic acids)
(Schumaker & Adams, 1969). These are
found in plasma and the four important
lipoproteins are chylomicrons, pre-
β-lipoprotein, β-lipoprotein and
α-lipoprotein. Hyperlipoproteinemia is
clinically significant, nowadays, as
lipoproteins are directly related with
atherosclerotic cardiovascular disease
(Arnao, Tuttolomondo, Daidone, &
Pinto, 2019).
1.3.3 Derived lipids
These are substances derived by hydrolysis
of simple or compound lipids.
 1.4 Proteins 11
FIGURE 1.6 (A) Cyclopentano perhydro phe-
nantherene ring system in steroids, and (B) molecu-
lar structure of cholesterol.

1. Steroids: These are abundantly found in carrier systems, such as liposomes, transfero-
nature. Steroids are derivative of complex somes, solid lipid nanoparticles, lipid nanos-
ring system named as cyclopentano tructures, etc.
perhydro phenantherene (Fig. 1.6A). The
important classes of steroids include sterols,
bile acids, sex hormones, adrenal cortical
hormones, Vitamin D, saponins and cardiac
1.4 Proteins
glycosides (Cole, Short, & Hooper, 2019).
Proteins constitute a diverse, heterogeneous
2. Sterols: Cholesterol is a well-known sterol
class of macromolecules and these may be said
(Fig. 1.6B). It is a white and waxy substance,
as the essence of life processes (Zaretsky &
widely distributed in all cells of the body,
Wreschner, 2008; Zhang et al., 2018). These are
particularly in nervous tissue. Cholesterol is
high molecular weight extremely complex
the precursor of bile salts, adrenocorticoids,
polymers of amino acids (Watford & Wu,
sex hormones, Vitamin D and cardiac
2018). In addition to carbon, hydrogen, oxygen
glycosides (Schade, Shey, & Eaton, 2020).
atoms, the molecular structure of proteins con-
Eicosapentaenoic acid (EPA) and
tains nitrogen and sometimes sulfur, phospho-
docosahexaenoic acid (DHA) play important
rus, iron, copper, manganese, iodine, zinc, and
roles with respect to immune functions,
other elements. The amino acids of proteins are
neurological and cardiovascular disorders
joined together with the help of peptide bonds
(Ochi & Tsuchiya, 2018).
(
CO
NH
). The common structure of pro-
Basic functions of different lipids are listed tein is presented in Fig. 1.7. These exhibit varie-
in Table 1.2. Recent studies have showed the ties of functions in cells by acting as structural
efficacy of various lipid-based drug delivery materials, carrier molecules, enzymes,

I. Background
12 1. Biological macromolecules: sources, properties, and functions

TABLE 1.2 Basic functions of lipids.

Type of lipids Functions

Fatty acids Precursor of triglyceride and source of energy

Triglyceride Storage of energy, thermal insulation and protection, binding of organs together
Cholesterol Component of cell membranes and also the Precursor of different steroids
Phospholipids Structural component of cell membranes and helps in digestion of fat
Bile acids Helps in fat digestion and absorption of nutrients
Eicosanoids Chemical messenger between cells

Dietary fat Carry lipid soluble Vitamins (A, D, E and K)

FIGURE 1.7 Common structure of protein.

lubricants, etc. (Zaretsky & Wreschner, 2008).
Proteins derived from different sources (ani-
mals and plants) have been used for the isola-
tion of peptides, and exhibited different
biological activities for humans (Daly et al.,
1990; Nayak, 2010). Proteins are classified in
three groups, namely simple proteins, conju-
gated proteins and derived proteins (Watford
& Wu, 2018).
1.4.1 Simple proteins
Upon hydrolysis, these types of proteins
yield only amino acids or their derivatives
(Murray, Harper, Granner, Mayes, & Rodwell,
2006; Watford & Wu, 2018). Simple proteins
are further classified into albumins, globulins,
glutelins, prolamins, albuminoids (scleropro-
teins), histones and protamines.
1. Albumin: These are soluble in water,
coagulated by heat, and precipitated by
saturated salt solution. Examples are
lactalbumin, serum albumin, egg albumin,
myogen of muscle, etc.
2. Globulin: These are soluble in dilute solution
of strong acids and bases and get coagulated
by heat. Examples are serum globulin,
ovoglobulin, myosin of muscle, etc.
3. Glutelin: These are soluble in dilute acids
and alkalis and get coagulated by heat.
Examples are glutenin from wheat and
oryzenin from rice, etc.

I. Background
 1.4 Proteins
4. Prolamines: These are soluble in 70%
80%
alcohol and insoluble in water, absolute
alcohol and other neutral solvents.
Examples are zein (corn), hordein (barley),
gliadin (wheat), etc.
5. Albuminoids (Screlroproteins): Albuminoids
are insoluble proteins and form supportive
tissues. These are animal proteins found in
hair, nails, horns and hooves. Examples are
keratin, collagen, gelatin, etc.
6. Histones: These are soluble in water, very
dilute acids and salt solutions. These are not
conjugated by heat and contain basic amino
acids. Examples include nucleic acids.
7. Protamines: These are the simplest of
proteins and are basic polypeptides, soluble
in water and ammonium hydroxides. These
are not conjugated by heat. Examples
include salmine (salmon sperm), clupeine
(herring sperm), etc.
1.4.2 Conjugated proteins
These contain simple protein combined with
nonprotein prosthetic group (Murray et al.,
2006). These include: (1) nucleoproteins (2) pro-
teoglycans and glycoproteins (3) chromopro-
teins (4) phosphoproteins (5) lipoproteins and
(6) metalloproteins (Murray et al., 2006;
Watford & Wu, 2018).
1. Nucleoproteins: These proteins are
composed of simple proteins with nucleic
acids as prosthetic group. In nucleoproteins,
protein moiety is usually a basic protein like
protamine and histone. Examples include
chromosomal proteins and some glandular
proteins.
2. Proteoglycans and glycoproteins: These
proteins contain carbohydrates as prosthetic
group like hyaluronic acid and chondroitin
sulpahte. These are mainly found in blood
plasma, gastric and salivary mucine,
immunoglobulins, human chorionic
gonadotropins, etc.
I. Background
13
3. Chromoproteins: These are composed of
simple proteins with chromotropic group as
prosthetic group. Examples include
hemoglobin (prosthetic group is heme),
flavoproteins (prosthetic group is
riboflavin), cytochrome (prosthetic group is
heme), etc.
4. Phosphoproteins: These are composed of
proteins and phosphoric acid as the
prosthetic group. Caesin (milk protein) and
vitelline (egg yolk protein) are the important
examples of this group.
5. Lipoproteins: These proteins are the
combination of proteins and lipids (fatty
acid, lecithin, cephalin, etc.) as prothetic
group. Lipoproteins occur in blood, cell
nuclei, milk, cell membranes, egg yolk, etc.
6. Metalloproteins: These are compounds of
proteins and some metals (such as iron,
cobalt, zinc, manganese, copper and
magnesium). Examples are ferritin,
ceruloplastin, carbonic anhydrase, etc.
1.4.3 Derived proteins
These are formed from simple and conjugate
proteins by denaturation or partial hydrolysis
(Murray et al., 2006). They are of two types (1)
denatured or primary derived proteins and (2)
secondary derived proteins (Murray et al.,
2006; Watford & Wu, 2018).
1. Denatured or primary derived proteins:
These proteins may be of different types.
a. Proteans—derived in the early stages of
protein hydrolysis by water, dilute acids
or alkalis or enzymes. Examples include
fibrin from fibrinogen, myosan from
myosin and edestan from edestin.
b. Metaproteins—derived by further
hydrolysis by stronger acids or alkalies,
which are insoluble in very dilute acids
and alkalis. Examples of such proteins
include acid metaproteins and alkali
metaproteins.
Another random document with
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had much use for honeymooning, but felt full of business.

"We had a carriage to ourselves by the kindness of the guard—a

Barnstaple man. And we talked. And when I got out of the train at Exeter, I
left her; and I've never seen her again and never shall. She was a stranger
woman to me for evermore."

He was silent for a time, but Dinah said nothing.

"It was her work, not mine. She'd got a dim sense of what she owed me,
I suppose, or else a fear of something. Yet, looking back, I often wondered
she troubled to tell me the truth, for she knew well enough I was much too
inexperienced and ignorant to have found it out. She might have lied.
Perhaps it was a case where a lie would have been best—if a lie's ever best.
Anyway it's to her credit, I suppose, that she told me. Not that she would
have done so if she'd known how I should take it. She reminded me of her
nest-egg and how I'd asked her how she came by it, and how she'd said an
uncle left it to her under his will. 'That's not true,' she said to me. 'And I
don't want to begin our married life with a secret between us, specially as it
happens to be such a trifle. I dare say some fools would pull a long face,'
she said, 'but you ain't that sort, else you'd never have fallen in love with
me.' Then she told me that for two years she'd been the mistress of a
gentleman at Bristol—a rich, educated man in business there. He'd kept her
till he was going to be married, and they parted very good friends and he
gave her a thousand pounds. He'd used her very well indeed and never
talked any nonsense about marrying her, or anything like that. It was just a
bargain, and he had what he wanted and so had she. Then she bent across
the carriage and put her arms round my neck and kissed me. But she kissed
a stone. I kept my head. I didn't go mad. I didn't curse or let on.

"I put her arms off me and bade her sit down and let me think; and all
the passion I felt against her kept inside me. I was man enough for that. She
looked a pretty thing that day. In pink she was, and if ever a man could
swear he looked at a virgin, he might have sworn it afore her grey eyes.

"I told her it was all up; and she kept her nerve too. A funny sort of
scene for any onlooker, to watch a newly-married man and woman starting
on their honeymoon and lost to all but a future bargain. Guard looked in and
had a laugh sometimes when the train stopped, and we ruled our faces and
grinned back at him.

"She began by trying hard to change me. She poured out a flood of
reasons; she used her quick brains as she'd never used them afore. But she
kept as keen and cool as a dealer to market, and when she found I wasn't
going on with it, she bided still a bit and then asked me what I was going to
do.

"That I couldn't tell her for the minute. 'Us'll begin at the beginning,' I
said, 'and have every step clear. You've got my name now, and you're my
wife in the law, and you've got your rights. And I shan't come between you
and them. But my love for you is dead. I don't hate you, because, I suppose,
women are mostly built like you and I won't waste my strength hating you.
You've gone. You're less to me now than the trees passing the window.
You'll live your life and I'll live mine,' I said to her; 'but you're outside mine
in future and I'm outside yours.'

"'That can't be,' she said. 'I've got a claim, and if you turn me down,
though I pray to God you won't—but if you do, you've got to think of my
future as well as your own.' I granted that and promised her she need not
trouble for herself. Being what I am, for good or evil, I saw very quick this
blow would fall on me, not her. She wouldn't miss me so long as everything
else was all right, and my feelings were such that I wasn't particular mindful
of myself, or my ruined hopes at that minute. I got a sudden, fierce longing
to cut a loss and be out of it. And that first driving impulse in me—to get
away from her and breathe clean air—stuck to me after twenty-four hours
had passed. Once knowing what she'd been, my love for her went out like a
candle. That may be curious, but so it was. I didn't fight myself over it, or
weaken, or hunger for her back. Never once did I. She was gone and
couldn't have been more gone if she'd dropped dead at my feet. All my
passion was a passion to get out of her sight.

"She tried with every bit of her cleverness to change me. Yes, she tried
hard, and I saw the wonder of her brains as I'd never even yet seen them.
She made a lot clear. She scorned the thing we call sin. She said to give a
man what she'd given was no more than to give another woman's baby a
drink from her breast if it was thirsty. She talked like that. She said she
never loved the man as she loved me, and she prayed very earnest indeed
for me to take a higher line and not be paltry. But it was all wind in the trees
for me and didn't shake me by a hair."

He stopped for a moment and Dinah asked him a question. She had
followed him word by word, her mouth open, her eyes fixed upon his face.

"If she'd told you before instead of after, would it have made a
difference?"

"Yes, it would," he said. "God's my judge, it would have made all the
difference between wanting her and loathing her. I'm the sort of man that
could no more have brooked it than I'd willingly touch a foul thing. That
may be silliness and a narrow understanding of life. Where women are
concerned, I may have wanted better bread than is made of wheat—I don't
know and I don't care; but that's me. And nothing could change me. She
tried hard enough—part for my own sake, I do believe, and part for hers.
She was wonderful and I'll grant it. She knew me well enough to waste not
a minute of her time in coaxing, or tears, or any foolery. She just kept to the
argument as close and keen as a man, and if she was feeling as much as me,
which ain't likely, she certainly didn't show it.

"She said a strange thing—bare-faced it seemed to me then, but I dare

say in strict fairness to her, I might have been shook by it. She reminded me
that it was what that blasted, rich man had taught her had made her what
she was. She said he'd lifted her above her class and woke up her brains and
educated her with books and lessons; and that what had drawn me was just
what she had to thank him for. She said, 'You'd never have looked at me
twice for myself. A pretty face means nothing to you. It was my sharpened
sense took you; and now you turn round and fling me off for just what made
you marry me.' Cunning as a snake she was—the wisdom and the poison
both. Or so it seemed to me. But what she said didn't alter the facts. Nothing
could alter them, and I wasn't built to take any man's leavings.

"She worked at me till we were very nearly to Exeter. Then she stopped
and said it was up to me to say what I intended. And I told her as to that she
needn't fear, because I'd do all that was right, and more. Her talk, you see,
had done this much. It made me understand that from her point of view—
hateful though it was—she had her rights. And so I bade her take her
luggage to one inn and I'd go to another; and next day I wrote to her that
she'd get a letter from me when I'd looked all round and decided what was
proper to do. She left me still hoping; I could see that. But she didn't hope
no more when she got my letter."

"You never went back on it?"

"Only once, for five minutes, that first night in bed, turning over my
future life and hers. For five minutes a thought did creep in my mind, and
for five minutes it stuck. It was such a thought as might have been expected
I dare say—a sort of thought any man might think; but it stank in five
minutes, and I shook it out. And the thought was how would it be if I said to
her she must give up her nest-egg and get rid of it for evermore, and then I
—— But what real difference did that make? None."

"Perhaps she wouldn't have let it go," said Dinah.

He nodded. It was another woman's view.

"Perhaps she wouldn't. She earned it—eh? Anyway the idea was too
dirty for me. Next morning I wrote and said what I was going to do. It was
pretty definite and that was where people said I was mad; but, looking back,
I can swear I'd do pretty much the same again. The thought was to be quick
—quick and away and out of it. Everything I'd done up to then tumbled
down that day. It was all gone together—not only her, but everything. I dare
say that was curious, but that's how I felt. I only asked for the clothes on my
back, and to get away in 'em and never see a bit of the past no more and
begin again."

"You'd feel like that."

"I did. I took a line she couldn't quarrel with. She made a fight; but
business was her god, and though I was a fool in her eyes, that didn't make
her inclined to play the fool. She hadn't to drive a bargain, or any such
thing. I cut the ground from under her feet, threw up the lot, handed her
over the business, lock, stock and barrel, and was gone, like a dead man out
of mind, so soon as I'd signed the proper papers."
 "She let you?"

"She couldn't do no otherwise, and as what I planned was well within

her sense of what was right and proper, she made no question. She pointed
out that she'd lost a good bit in any case with a mystery like this hanging
over her; and she also wrote, when all was fixed up, that she hoped I'd live
to change my mind and come back to her and very thankful she would be if
I was to. I dare say she truly thought I would.

"We were in Exeter for a week and came and went from a lawyer's—but
never there together. I ordained to give her what I'd got and leave her to do
as she pleased. She was sorry I saw it like that; but the sense of the woman
never allowed nothing to come between her and reason. The lawyer tried to
change me too. He was a very kindly man. But it went through. She took
over the dairy and carried on my engagements to my aunt, and no doubt
developed the shop same as I meant to. She gave out I'd gone away for a bit
and might be back in a month. I don't suppose anybody ever heard more,
and when I didn't come back, she had a search made for me all very right
and regular; but I'd gone beyond finding, and she carried on; and no doubt
the nine days' wonder died in course of time. Only my aunt knew I'd gone
of my own accord; but why I'd gone, only one creature beside my wife ever
knew; and that was her mother; and I doubt not she sided with her daughter.
I dare say there's a lot more the other side could tell; but I made a clean cut.
I dropped every creature and began again out of their reach. That's the story
of me, Dinah. I've most forgotten many of the details myself now. It's seven
and a half years agone. I saw in a North Devon paper my old aunt was dead,
and so Minnie's free of them payments and standing alone. Half my savings
she had also."

There was silence between them for more than a minute. Then Dinah
spoke, went back to his first word and asked a question.

"D'you call that being married?"

"Yes—that's my marriage. There ain't much more to tell. I was for going
to Canada, and started unknown with fifty pounds of money, which was all
I kept. I was going to get a state-aided passage from London and begin
again out there. It sounds a big thing to fling over the whole of your life,
like as if you was taking off a suit of old clothes; but it didn't seem big to
me then—only natural and proper. I comed even to like it. But chance
willed different, and the accident of meeting a stranger in the train kept me
in England after all. Chance done me a very good turn then. A farmer got in
the train at Taunton and between Taunton and Bath, fate, or what you like to
call it, willed I went to that man. We got talking, and I told him I was going
abroad, being skilful at cows and the butter and milk business. He got
interested at that and reckoned I might be such a man as he needed; but I
said plainly that I was cutting losses, and my past must bide out of sight,
and I'd best to go foreign in my opinion. By that time, however, he'd got a
fancy he'd trust me. He was a very good man and a judge of character,
which most good men are not in my experience. I found after that he was a
rare sort of chap—the best and truest friend to me—such a man as inclined
me slowly to think the better of the world again. He only asked me one
question and that was if, on my honour, I could tell him I'd done no
dishonest or wicked thing from which I was trying to escape. And I swore
by God I had not. He believed me, and when, a day or two later, I told him
the whole story, he didn't say whether in his judgment I'd done right or
wrong, but he granted that I'd done right from my point of view and thought
no worse of me for it. I hesitated a bit at his offer; but I liked him,
somehow, from the first, and I was cruel tired, and the thought of getting to
work right away was good to me. Because I knew by then that there was
nothing like working your fingers to the bone to dull pain of mind and make
you sleep.

"My life with him is another tale. I look back upon it with nothing but
content. I did well by him, and he was as good as a father to me. It's near
eighteen months ago he died, and his two sons carried on. Very nice men,
and they wanted me to stop; but I couldn't bide when the old chap dropped
out. He left me two hundred pounds under his will, Dinah; and his sons
didn't object that I should take it, for they were well-to-do and liked me.
Then I saw Joe's advertisement in the paper and had a fancy to come back
alongside where I was born."

"And Mrs. Maynard never found you?"

 "No; but she isn't Mrs. Maynard. Maynard's not my name and Lawrence
ain't my name."

She sighed.

"Man!" she said, "you be sinking and sinking—oh, my God, you be

sinking out of my sight! I thought you was one creature, and now you be
turning into a far-away thing under my eyes."

"I don't feel like that. I'm Lawrence Maynard to myself, Dinah. T'other
be dead and in his grave. My name was Courtier. There's some of the family
about on Dartmoor yet. My great-grandfather was a Frenchman—a soldier
took in the wars more than a hundred year ago. And the moor folk traded at
the war prisons to Princetown, so he got to know a good few at prison
market. Then he was tokened to a farmer's daughter, and after the peace he
married her and stopped in England and started a family."

"What's your other real name then?"

"Gilbert, same as my father."

"Us must be going," she said.

"Shall I tell her to hot some more tea for you?"

"No—I don't want no tea."

He drank his cold cup at a draught and pressed her to eat a little; but she
shook her head.

"I'll see you home by New Bridge and then get up back through the
woods, Dinah."

"I can travel alone."

"No, you mustn't do that."

She said very little during the long tramp through a night-hidden land.
The darkness, the loneliness, the rustle of the last dead leaves and the
murmur of the wind chimed with her thoughts. She seemed hardly
conscious of the man at her side. He strove once or twice to talk, but found
it vain and soon fell into silence. At New Bridge Dinah spoke.

"You'll always be 'Lawrence' to me," she said. "Tell me this. When are
you going to see me again, after I've thought a bit?"

"Like you to want to. We can meet somewhere."

"You love me?"

"Yes; as I never thought I could love anything. But how should you love
me any more?"

She did not answer immediately. For some distance they walked by the
river. Then they reached a fork of the road where their paths divided; for
here Dinah climbed to the left by a steep lane that would bring her to Lower
Town and home, while Maynard must ascend into the woods.

They stopped.

"Will you do this?" she said. "Will you put the story of your life before
Enoch Withycombe?"

"Why, Dinah?"

"To get his opinion on it—all—every bit."

"Yes, if you like."

"I do like. I'm very wishful to know what a man such as him would
say."

"If he's well enough, I'll see him to-morrow. It's been in my mind to tell
him about myself before to-day."

"I wish you had."

 "He shall hear it. I set great store by his sense. He might—— Can you
get home from here? I'll come with you if you like."

"No."

"You've forgiven me?"

"I'll think and think. Be there anything to forgive?"

"I don't know. And yet I do. Yes—you think—then you'll find you've
got to forgive me for ever loving you, Dinah."

"You're life—you're life to me," she said. "Don't say small things like
that. I'm only being sorry for all you've had to suffer all these years and
years. I'll go on being sorry for you a long time yet. Then I'll see if I'm
angry with you after. I can only think of one thing at a time."

She tramped up the hill and he stood, until her footfall had ceased. Then
he went his own way and had climbed to within half a mile of Buckland,
when a strange thing happened. He heard the winding of a hunter's horn.
Through the darkness, for all listening ears at Holne or Leusden, Buckland
or the neighbour farms and hillsides to hear, came the melodious note. It
rang out twice, clear and full; and kennelled hounds a mile distant caught it
and bayed across the night—a farewell, good to the heart of Enoch
Withycombe if he had heard them.

CHAPTER XXI

FUNERAL

Enoch Withycombe had always promised to sound his horn again in

sight of his end, and three days after he woke the echoes of the Vale he
died. On the night that his music vibrated over hill and valley for the last
time, Melinda had pushed his chair to the cottage door. When Lawrence
called on the following Sunday afternoon, though he sat for a while beside
his bed, the old hunter had already drifted into a comatose state, and the
story Maynard had hoped to tell was never heard by him.

A bitter grey day dawned for a funeral attended by unusual mourners.

The dead sportsman's master had made a promise and he kept it. Hounds
did not meet that day; but the master, the huntsman and the whipper-in both
clad in pink, and two brace of hounds were at the grave side—a bright flash
of colour in the sombre little crowd that assembled.

Melinda Honeysett and her brother, Jerry, were chief mourners, while
behind them came the fox-hunters; and of those who followed, some took it
amiss to see such an addition to a funeral; while others held it most seemly
and fitting.

Indeed for many days afterwards the question was heavily debated, and
Arthur Chaffe and Ben Bamsey, who were both at the grave side,
considered squire and parson alike to blame for an impropriety; while Joe
Stockman, who came with Susan, Maynard and Thomas Palk, highly
approved of the innovation. John Bamsey and Lawrence were among the
bearers. They had also helped to carry the dead man from his home to the
grave, for it was a walking funeral. Half a dozen private carriages followed
it, and Melinda was bewildered to arrange the many gifts of flowers that
came to her from her father's old friends of the countryside.

"Fox-hunters have long memories seemingly," said Jerry to his sister, as

they read the cards attached to wreath and cross.

After the funeral was ended and when Enoch lay beside his wife, on the
north of the church tower beneath a naked sycamore, it happened that
Maynard found Dinah Waycott beside him in the press of the people. She
had come with the Bamseys and, knowing that he would be there, now
reached his side, bade him "good day," and unseen put a letter into his hand.

For a moment he picked up the thread of their conversation, where they

had left it on the night by Dart River a week before.
 "I couldn't tell him—he was too far gone next day," he said quietly,
taking her letter.

"No matter," she answered, and then moved away.

The crowd drifted down the lanes and up the lanes. The men in pink
mounted their horses and rode away with the hounds. Enoch's old master
also departed on horseback, as did a dozen other men and several women.
Soon only Melinda and Jerry were left to see the grave filled in and dispose
the wreaths upon it. Mr. Chaffe kept them company. He cheered them by
saying that never in his long experience, save once, had he known any man
of the people enjoy such splendid and distinguished obsequies.

"A magnificent funeral despite the hounds," he said, "and Buckland did
ought to be proud of it. There was a journalist from a Plymouth newspaper
there, Jerry, so you'll be able to keep a printed history, with all the names,
for future generations of your family to read aloud."

But Jerry was weeping and paid no heed; while his sister also, now that
the strain had passed and the anticlimax come, hid not her tears.

Soosie-Toosie, her father and the two labouring men walked home
together and Joe uttered a vain lament.

"A thousand pities the man's sailor son, Robert, couldn't be there," he
said. "It would have been a fine thing for him to see what his father was
thought of. And he'd have supported Melinda. She stood up very well and
firm; but I know she'll miss him a terrible lot—her occupation gone you
may say; for there's nobody leaves such a gap as an invalid that's called for
your nursing for years. When the place is suddenly emptied of such a one,
you feel as if the bottom was knocked out of your life, same as I did when
my wife went."

Joe was in a mood unusually pensive and his daughter felt anxious. She
tried to rally him, but failed.

"I'm looking forward," he said. "In that great rally of neighbours there
was a lot of old blids from round about—a good few up home eighty years
old I shouldn't wonder; and such was the bitter cold in the churchyard that
you may be certain death was busy sowing his seeds. I hope to God I be all
right, and I thank you for making me put on my heavy clothes, Soosie."

Palk walked behind them and talked fitfully to Maynard.

"'Twill ruin Christmas," said Thomas. "He was a famous man and
there'll be a gloom fall over the place now he's dropped out."

"It won't make any difference," answered the younger.

"It may make a valiant lot of difference, and that nearer home than you
think for," answered Palk.

But Maynard shook his head.

"There's nothing in it. Joe won't offer for her—Mrs. Honeysett—if that's
what you're thinking; and if he did, 'tis doubtful if she'd take him. I've heard
her tell about him to her father."

"And what did she tell?"

"Nothing but good. She knows his worth and all that. But Enoch didn't
set very high store on master. I wondered why sometimes."

"Did you? I lay he knew him better than what you do. And he knew this
—that a man who worked his only child like Stockman works his would
make his wife a proper beast of burden."

"Everybody's selfish. I dare say when the news of the rise reaches us
presently, you'll think better of him."

Then Stockman called Lawrence and Susan fell back to the horseman.

"He wants to tell Maynard about some ideas he's got, and it will distract
his mind to do so," she explained.

"Be master under the weather about Mr. Withycombe, or is he only

pretending?" asked Thomas bluntly.
 "He's a very feeling creature is father," answered the woman. "He didn't
care much for poor Mr. Withycombe, and Mr. Withycombe never quite saw
father's good points, like most of the people do; but father's down-daunted
to-day. 'Tis a landmark gone; and death's death; and he's fearful that another
old person here and there may be took presently, along of the cruel cold in
the churchyard."

"The wind curdled down off the Beacon like knives," admitted Palk.
"Mrs. Honeysett kept her face very steady."

"She did. But she's a brave creature."

"She've got the cottage for her life, however."

"Yes. Squire's left it to her for naught, so long as she likes to bide there."

"A deep thought—how long she will bide there."

"Yes, it is. Jerry will be gone, come presently; but she'll have a
neighbour. There's a widow man and his daughter took the cottage—the
haunted house that joins hers. He's a new gardener to Buckland Court and
don't fear ghosts."

"So I heard tell."

They were silent and then Thomas, now on very friendly terms with
Susan, asked a question.

"Will it make a difference to Mr. Stockman, Mrs. Honeysett being set

free of her father, miss?"

"I couldn't tell you, Tom. I've axed myself that question. But I'm not in
father's thoughts."

His caution made him hesitate to speak again, but he knew that another
question would go no farther than his listener.

"And if I may venture to put it, would you like to see him wed, miss?"
 Susan slowed her steps that no sound of their voices might reach Joe.
Her eyes were on his back as she answered.

"Yes, I think I would. A wife would add to his peace and comfort."

"She might add to yours."

"She might; but I'm not troubling as to that. Still, if she was a nice
woman, I dare say she would."

"A wife—nice or otherwise—would open your father's eyes," declared

Thomas. "In all respect I say it; but where you be concerned, he's got to
make such a habit of you, and got to take you so terrible much like he takes
his breakfast, or his boots, or any other item of his life, that it would be a
very good thing for his character if he found out what you was."

"He don't undervalue me I hope," answered Susan. "Because a man

don't say much, it don't follow he don't feel much, Thomas."

"But he do undervalue you cruel, and for that reason I'd be very pleased
indeed if he was to get a woman for himself. Because no female he'm likely
to find will show your Christian power of taking everything lying down. In
fact no woman as ever I heard tell about can rise to such heights in that
partickler as you; and your father have got so used to you, like a good pixy
about the place, ready and willing to work night and day; and if he was up
against another woman, he'd very soon have the surprise of his life."

"If a wife was so fond of him as what I am, she'd treat him so faithful as
what I do," argued Soosie-Toosie; but Thomas assured her that she was
mistaken.

"Don't think it," he said. "No wife ever I heard tell about would drudge
for nought same as you. However, I be going beyond my business, and no
doubt you'll tell me so. But 'tis only on your account, I assure you."

"I know it, Tom, and I thank you for your good opinion. But father's
built in a higher mould than you and me. He's born to command, and I'm
born to obey. Us generally do what's easiest, to save trouble; and if he was
to marry again, he'd still be born to command, and any woman, knowing
him well enough to take him, would understand that."

"They might, or they might not," argued Mr. Palk. "When a man goes
courting, he hides a lot in that matter and, strong though the governor may
be, there's women very well able to hold their own against any man born;
and Melindy Honeysett is one. But it may happen. The mills of God may be
grinding for it; and then master would look at you, and the scales would fall
from his eyes I expect."

As soon as he was alone, Lawrence Maynard read the letter from Dinah.
It was the first time he had ever seen her writing, and he found it a large,
free hand with a hopeful slope upwards at the end of each line.

But the note was very brief. She committed herself to no opinions and
only begged Lawrence to come to her in Lizwell Woods, a mile or two from
her home, on the following Sunday afternoon.

"I'll be where the Webburn rivers run together, so soon after three
o'clock as I may," she said.
 CHAPTER XXII

AT WATERSMEET

Dinah was first at the tryst and doubted not that Maynard would come.
The lonely, naked woods swept round her and she sat on a fallen trunk not
far from where the Webburn sisters shot the grey forest with light and
foamed together beneath the feet of trees. The day was dull and windy with
rain promised from the south. Withered beech leaves whirled about Dinah's
feet in little eddies, then rushed and huddled away together in hurtling
companies—with a sound like a kettle boiling over, thought Dinah. Her
mind was not wholly upon Maynard, for Joe Stockman's gloomy prophecy
had come true in one case and Mr. Bamsey was indisposed from a chill
caught at the funeral. As yet they were not concerned for him; but he had
grown somewhat worse since the preceding day and Faith had sent Jane to
fetch the doctor. Jane never declined a commission that would take her into
Ashburton.

A smudge of black appeared in the woods and Maynard stood on the

east bank of the river. Dinah rose and waved to him; then he ascended the
stream until a place for crossing appeared. Here he leapt from stone to stone
and was soon beside her. They wandered away and he found a spot
presently, where the ground was dry with fallen needles from a pine above
it.

"Sit here," he said, "a little while."

She had not spoken till now, save to tell him her foster-father was ill.
But when they sat side by side, with the bole of the great pine behind them
and its lower boughs sweeping about them to the ground, she answered all
the questions he wanted to put in one swift action. For a moment she looked
at him and her face glowed; and then she put her arms round his neck and
kissed him.

"Dinah—d'you mean it?" he said. "Oh, d'you mean all that?"

 "I want you; I can't live my life without you, Lawrence."

"After what I've told you?"

His arms were round her now and he had paid her fiercely for her kiss.

"What is marriage? I've been puzzling about it. I've been puzzling about
it for years, for it seems years since you told me you was married. And if
you knew what I'd been feeling, or how I fought not to kiss you at the
funeral, you'd be sorry for me. But you've only been sorry for yourself I
expect, you selfish man."

He did not answer. He had released her, but was still holding one of her
hands.

"I'd make you a good wife, Lawrence," she said.

"By God you would!"

"And what is marriage then? Why d'you tell me you're married to her—
any more than I'm married to John Bamsey—or anybody?"

"Marriage is a matter of law, and a man can only marry one wife."

"And what's a wife then?"

"The woman you are married to—she that's got your name."

"Would you say your wife was married?"

"Certainly she is."

"A widow then?"

"Not a widow if her husband is alive."

"Then why d'you say that Gilbert Courtier died when Lawrence
Maynard came to life? If Gilbert Courtier's dead, then his wife is a widow."
 Her literal interpretation was not a jest. He perceived that Dinah
presented no playful mood. She was arguing as though concerned with
facts, and not recognising any figurative significance in what he had told
her about himself. For a moment, however, he could hardly believe she was
in earnest.

"If it was as easy as that," he said.

"How d'you feel to it then?"

"I feel to it as you do, with all my heart. God knows what I want—one
thing afore all things and above all things: and that's to have you for my
own—my own. And whether I can, or can't, my own you will be from this
hour, since you want to be my own, Dinah."

"And I will have it so. You're my life now—everything."

"But you can't make me less than I am. It's no good saying that Gilbert
Courtier's dead; and though I change my name for my own comfort, that's
not to change it against the facts."

"D'you want to go back to it then?"

"Not I. I'll never go back, and 'tis no odds to me what I'm called; but a
wife's a wife, and my wife must stand safe within the law—for her own
safety—and her husband's honour."

She stared at this.

"D'you feel that?"

"I do, Dinah."

"That things like safety, or the law, matter?"

"To you—not to me."

"What do I know about the law—or care? D'you think I'm a coward?
You've only got one name for me, and ban't the name I love best in the
world good enough? Who else matters to you, if you're Lawrence Maynard
to me? And what else matters to you if I love you? Words! What are words
alongside the things they stand for? I want you, same as you want me. And
whose honour's hurt?"

"You feel all that?"

"Not if you don't. But you do."

His own standards failed for the time and he said somewhat more than
he meant. Such love as Dinah's, such certainty as Dinah's, made doubt, built
on old inherited instincts, look almost contemptible. Trouble of old had
shaken these deep foundations; now happiness and pride at his splendid
achievement similarly shook them.

"Yes I do," he said. "There's naught else on God's earth; I'd let all go
down the wind afore I'd lose what I've won. I can keep off words as easy as
you; and the word that would come between me and such love as I've got
for you was never spoke and never will be. Words are dust and can go to the
dust. But——"

He had recollected a fact beyond any power of words to annul.

"There's a hard and fast reality, Dinah, and we've got to take it into
account, for it can't be argued down, or thought away."

"Then let it go—same as everything else have got to go. There's only
one thing matters, I tell you, and we feel the same about it. Love's far too
strong for all other realities, Lawrence. There's only one reality: that you
and me are going to live together all our lives. What fact can stand against
that? If facts were as big as the Beacon, they're naught against that fact. You
be my own and I'm your own, and what else signifies?"

"You make me feel small," he said, "and love so big as that would make
any man feel small, I reckon. And for the minute I'll put away the ways and
means and machinery, that always have to be set running when a man wants
to wed a woman."
 "What's machinery to us? We didn't love each other by machinery and
us shan't wed by machinery."

"Us can't wed without machinery."

"You say that! Ban't us wed a'ready? Be the rest of it half so fine as
what brought us together, and made us know that our lives couldn't be lived
apart? Ban't you wed to me, Lawrence?"

"I am," he said, "and only death will end it. But there's more than that
for you; and so there's got to be more for me. And if I'm going to be small
now and talk small, it's for you I do, not for myself. You're a sacred thing to
me and holy evermore mind."

"And you be sacred to me," she said. "You've made all men sacred and
holy to me; and you've made me feel different to the least of 'em, because
they be built on the same pattern as you. I swear I feel kinder and better to
everybody on earth since I know you loved me so true."

"It's this, then—a bit of the past. When I first came here I felt, somehow,
that in Stockman I'd had the good luck to hit on just such another as my old
master up country. He seemed to share the same large outlook and
understanding, and I found him a man so friendly and charitable with his
neighbours that I told him about myself, just like I told the other; and he
was just the same about it—generous and understanding. In fact, he went
further than my old master, and agreed with me right through, and said it
was a very manly thing to have done, and that if more people had the pluck
to cut a loss, the world would go smoother. He praised me for what I'd
done, and I remember what he said. He said, 'To let sleeping dogs lie be a
very wise rule; and to let sleeping bitches lie be still wiser.' But I know a lot
more about Joe Stockman now than I did then; and though I've got no
quarrel with him, yet, if the time was to come again, I wouldn't tell him.
He'd never tell again, or anything like that; but he knows it, and if I was to
say to him that I held I wasn't married and wanted another, he'd laugh at
me."

Dinah admitted that Mr. Stockman was a serious difficulty.