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Filedate - 468download Ebook Biological Macromolecules Bioactivity and Biomedical Applications PDF Full Chapter PDF
Filedate - 468download Ebook Biological Macromolecules Bioactivity and Biomedical Applications PDF Full Chapter PDF
Edited by
Dilipkumar Pal
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur, India
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v
vi Contents
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
xiii
xiv List of contributors
The scope of this book, entitled Biological This book, containing 4 sections and 26
Macromolecules: Bioactivity and Biomedical chapters, provides a systematic insight into the
Applications, is the coverage and review of inclusive discussions on bioactivity and bio-
recent trends and applications of biological medical applications of different biological
macromolecules, such as carbohydrates, lipids, macromolecules. We are glad to see that many
proteins, peptides, and nucleic acids in biome- authors across the globe accepted our invita-
dicines, drug delivery, growth factors delivery, tion and contributed valued chapters for this
nutrients and nucleic acids delivery, cell encap- book, covering a wide spectrum of fields. A
sulation, enzyme mobilization, and tissue concise account of the contents of each chapter
engineering. has been described to provide a glimpse of the
The mysteries of life lie in biological macro- book to the potential readers of various fields.
molecules. A large volume of biological macro- The topics in the book (in order of prefer-
molecules is obtained from different biological ence) include the following: Biological
origins such as plants, algae, fungi, animals, Macromolecules: Sources, Properties, and functions
and microbial sources. Biological macromole- (Chapter 1)—this chapter describes sources
cules exhibit some significant and favorable physicochemical properties, bioactivity and
advantages over synthetic macromolecules, biomedical applications of different biological
such as sustainable and economic production, macromolecules concisely; Structure Activity
biocompatibility, biodegradability, and Relationship of Biological Macromolecules
improved bioavailability. In recent years, a (Chapter 2)—this chapter aims to provide an
plethora of biological macromolecules (carbo- overview of the structural features influencing
hydrates, lipids, proteins, peptides, and nucleic the bioactivities of biological macromolecules,
acids) has been used in the biomedical and namely L-amino acid oxidases, lysostaphin,
healthcare fields. They showed varieties of and metallo-β-lactamase-such as lactonase and
bioactivities such as antioxidant, anticancer, chitosan; The Importance of Biological
antidiabetic, antimicrobial, immunomodula- Macromolecules in Biomedicine (Chapter 3)—this
tory activities on the central nervous system, chapter highlights the therapeutic aspects of
and gastrointestinal activity. The other biomed- macromolecules and the medicinal use of bio-
ical applications include drug delivery, growth logical macromolecules against various dis-
factors delivery, nutrients and nucleic acids eases and ailments; Modification Techniques for
delivery, cell encapsulation, enzyme mobiliza- Carbohydrate Macromolecules (Chapter 4)—this
tion, and tissue engineering. The structure- chapter characteristically abridges the signifi-
property relationship is also an important cant developments of the last five to ten years
aspect for a thorough understanding of the bio- and discusses critically in the area of modifica-
activity of biological macromolecules. tion of carbohydrates macromolecules;
xix
xx Preface
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
Biological Macromolecules
DOI: https://doi.org/10.1016/B978-0-323-85759-8.00005-1 3 © 2022 Elsevier Inc. All rights reserved.
4 1. Biological macromolecules: sources, properties, and functions
(Corn, Windham, & Rafat, 2020; Oana, (Hasnain et al., 2020; Kandar, Hasnain, &
Adriana, Mircea, Dragos, & Monica, 2018; Nayak, 2021; Maity et al., 2021; Nayak &
Rodrigues Mantuano, Natoli, Zippelius, & Hasnain, 2019a, 2019b; Nayak, Hasnain, Dhara,
Läubli, 2020; Sun, Jing, Ma, Feng, & Hu, 2020). & Pal, 2021; Pal, Saha, Nayak et al., 2019).
Another important area of research is immuno- Polysaccharide and proteins are used exten-
modulators with respect to present SARS-CoV-2 sively for the preparation of hydrogels for
perspective, where the biological macromole- drug delivery, tissue regeneration, wound
cules, mainly proteins, play significant role (Ji dressings, etc. (Del Valle, Dı́az, & Puiggalı́,
et al., 2020). Proteins are associated with the 2017; Nayak & Pal, 2016b; Nayak, Hasnain,
development process of immune system (Daly, Pal, Banerjee, & Pal, 2020; Pal, Nayak, & Saha,
Reynolds, Sigal, Shou, & Liberman, 1990). 2019a, 2019b; Ray et al., 2020). Beside drug
Lipids are also responsible to play key role as delivery, the biological macromolecules have
adjuvants for the development of vaccines continuously been used to formulate delivery
(Martinez-Gil, Goff, & Tan, 2018; Schwendener, carrier-systems for growth factors (Rao, Rekha,
2014). Day by day, the incidence of lifestyle Anil, Lowe, & Venkatesan, 2019; Shariatinia,
diseases more specifically diabetes, hyperten- 2019). Polysaccharides are also employed for
sion, etc., are increasing vertically, where the the encapsulation of various bioactive sub-
roles of biological macromolecules have been stances like vitamins and nutraceuticals (Bala,
studied and were found to be utilized widely Singha, & Patra, 2019; Lauro, Amato, Sansone,
as antidiabetic agents (Alam, Shafique, Amjad, Carbone, & Puglisi, 2019). The current chapter
& Bin Asad, 2019; Hu, Nie, & Xie, 2018; Rı́os, deals with a brief discussion about the sources,
Francini, & Schinella, 2015; Yu, Shen, Song, & properties, and valuable applications of vari-
Xie, 2018). They cause increase in insulin secre- ous biological macromolecules like carbohy-
tion and thus, reduce the blood glucose level drates, lipids, proteins and nucleic acids.
(Rı́os et al., 2015). Chitosan is a well-known
polysaccharide, which is reported to exhibit
antimicrobial and antidiabetic activities
(Hasnain & Nayak, 2018; Karadeniz & Kim, 1.2 Carbohydrates
2014; Rabea, Badawy, Stevens, Smagghe, &
Steurbaut, 2003). Extensive research is going The most widely found organic compounds
on in the area of tissue engineering for the in nature are carbohydrates. These are well-
development of artificial tissue to repair and known as very essential source of life or sus-
replace defective or diseased tissue or organs taining life itself (Slavin & Carlson, 2014).
(Hasnain, Ahmad, Chaudhary, Hoda, & Carbohydrates are commonly found in plants,
Nayak, 2019; Nayak, Ahmed, Tabish, & microorganisms and animal tissues (Werz &
Hasnain, 2019; Pal, Saha, Nayak, & Hasnain, Seeberger, 2005). These are also present in
2019). Naturally derived biological macromole- blood, tissue fluids, etc. (Kilcoyne & Joshi,
cules like chitosan, alginate, carrageenan, 2007). Carbon, hydrogen and oxygen are the
ulvan, gelatin, etc., have been used for three primary elements of molecular structure
bone tissue regeneration (Hasnain, Nayak, of carbohydrates (Luo et al., 2020; Werz &
Singh, & Ahmad, 2010; Maity, Hasnain, Nayak, Seeberger, 2005). These are optically active
& Aminabavi, 2021; Nayak, Ahmed, Tabish, & polyhydroxy aldehydes or ketones. There are
Hasnain, 2019). Numbers of polysaccharide three major classes of carbohydrates, broadly,
have long been used in different types of drug monosaccharides, oligosaccharides and poly-
delivery systems as biopolymeric excipients saccharides (Slavin & Carlson, 2014).
I. Background
1.2 Carbohydrates 5
I. Background
6 1. Biological macromolecules: sources, properties, and functions
animals, starches are the most important properties like low density, flexibility, high
source of food. These consist of two types of strength, biocompatibility, biodegradability,
molecules (Fig. 1.1): (A) linear and water etc., which suggest for biomedical
soluble component, e.g., amylose and (B) applications (Hasnain et al., 2020; Kandar
branched water insoluble, e.g., amylopectin. et al., 2021). In traditional healthcare,
Commercially, starch is produced mostly cellulosic biomaterials play an important role
from corn, but wheat starch, potato starch, and recently, some significant areas of
and tapioca starch are also used (Nayak application are being explored with the uses
et al., 2020). of cellulose like drug delivery (Hasnain et al.,
2. Cellulose—It is the chief constituent of 2020; Kandar et al., 2021; Pal et al., 2019b),
fibrous parts of the plants and consequently, tissue engineering (Hasnain, Nayak, Singh, &
is the most abundant organic material Ahmad, 2010; Murizan, Mustafa, Ngadiman,
occurring in nature (Pal et al., 2019b). It is Mohd Yusof, & Idris, 2020), management of
made up of long chains of β D-glucose wound (Alven & Aderibigbe, 2020), etc. By
molecules linked by 1, 4-linkages (Fig. 1.2). It using quaternary ammonium salt the surface
serves as bulk forming agent of the food. of cellulose nanocrystals (CNC) is modified,
Undigested cellulose increases the bulk of which can inhibit the growth of Staphylococcus
feces and helps in the evacuation of bowels. aureus and Escherichia coli (Tavakolian, Jafari,
Cellulose exhibits some of important & van de Ven, 2020).
FIGURE 1.1 Molecular structure of starch: (A) linear and water soluble component: amylose and (B) branched water
insoluble: amylopectin.
I. Background
1.2 Carbohydrates 7
FIGURE 1.2 Molecular
structure of cellulose.
I. Background
8 1. Biological macromolecules: sources, properties, and functions
acid (G-unit) (Hasnain et al., 2020; Kandar used in the biomedical applications mainly
et al., 2021; Nayak et al., 2021). Molecular in drug delivery and tissue regeneration
structure of pectin is presented in Fig. 1.5. It (Malakar, Nayak, Jana, & Pal, 2013; Malakar,
is processed from marine algae and giant Nayak, & Das, 2013).
kelp as raw materials. These are widely 7. Chitosan—It is a cationic natured
used as thickener, emulsifier, stabilizer, etc. carbohydrate polysaccharide, extracted by
(Kandar et al., 2021). Alginate based drugs deacetylation of chitin (Hasnain & Nayak,
are effectively used for antimicrobial as well 2018). It is reported to show various
as antiviral therapy (Szekalska, Pucilowska, biological properties including antidiabetic,
Szymańska, Ciosek, & Winnicka, 2016). The antioxidant, immune-enhancing,
alginate film with EDTA exhibited stronger antimicrobial as well as anticancer activities
antimicrobial effects against Gram-negative (Hasnain & Nayak, 2018; Rabea et al., 2003;
bacteria, especially, in case of processed Rı́os et al., 2015). Chitosan is very much
food packaging (Senturk Parreidt, Müller, & effective in the formulation of insulin with
Schmid, 2018). Structural modifications of controlled delivery functionality at the target
alginates can easily be made by using site (Barbosa et al., 2020). Carboxymethyl-
crosslinkers, improvise the mechanical hexanoyl derivative and polyethylene glycol-
strength and cell affinity and was widely trimethyl complexes of chitosan have been
I. Background
1.3 Lipids 9
found to possess fat-lowering and fat- 9. Glycogen—This is also known as animal
preventing properties. Chitosan-based starch, as it is a principal polysaccharide
collagen complex sponges showed occurring in animal tissues, specifically in
effectiveness in the healing of diabetic liver and muscle (Roach, Depaoli-Roach,
wounds (Wang et al., 2008). Chitosan and its Hurley, & Tagliabracci, 2012). Glycogen is
derivatives are being extremely used in many the storage form of energy release, quickly,
biomedical applications, such as drug when needed (Kreitzman, Coxon, & Szaz,
delivery, tissue engineering, wound dressing, 1992). Similar to starch, this is also
orthopedics, etc. (Hasnain et al., 2020; composed of glucose units united by 1, 4-
Hasnain, Ahmad, Chaudhary, Hoda, & linkages and branches arising by 1, 6-
Nayak, 2019; Hasnain, Nayak, Singh, & linkages.
Ahmad, 2010; Kandar, Hasnain, & Nayak,
Sources and functions of various polysac-
2021; Maity, Hasnain, Nayak, & Aminabavi,
charides are listed in Table 1.1.
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 1.3 Lipids
ester of a complex galactose polysaccharide
(Kandar et al., 2021). It is nondigestible Lipids are heterogeneous group of organic
material and is used as a bulk laxative. compounds, related either actually or poten-
Recent years, a number of biocompatible tially, to the fatty acids (Pandey & Kohli, 2018).
agar-based composite has been formulated They are poorly soluble in water and soluble in
for their potential applications in biomedical nonpolar solvents like chloroform, benzene,
fields including drug delivery and tissue petroleum ether, etc. In our body, lipids are an
engineering applications (Kandar et al., integral part of the cell membrane structure,
2021; Nayak, Alkahtani, & Hasnain, 2021; metabolic fuel and storage form of energy (van
Shah et al., 2019). Meer et al., 2008; Zheng et al., 2019). Lipids act
I. Background
10 1. Biological macromolecules: sources, properties, and functions
I. Background
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).
(CONH). 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
lubricants, etc. (Zaretsky & Wreschner, 2008). are further classified into albumins, globulins,
Proteins derived from different sources (ani- glutelins, prolamins, albuminoids (scleropro-
mals and plants) have been used for the isola- teins), histones and protamines.
tion of peptides, and exhibited different
1. Albumin: These are soluble in water,
biological activities for humans (Daly et al.,
coagulated by heat, and precipitated by
1990; Nayak, 2010). Proteins are classified in
saturated salt solution. Examples are
three groups, namely simple proteins, conju-
lactalbumin, serum albumin, egg albumin,
gated proteins and derived proteins (Watford
myogen of muscle, etc.
& Wu, 2018).
2. Globulin: These are soluble in dilute solution
of strong acids and bases and get coagulated
by heat. Examples are serum globulin,
1.4.1 Simple proteins ovoglobulin, myosin of muscle, etc.
Upon hydrolysis, these types of proteins 3. Glutelin: These are soluble in dilute acids
yield only amino acids or their derivatives and alkalis and get coagulated by heat.
(Murray, Harper, Granner, Mayes, & Rodwell, Examples are glutenin from wheat and
2006; Watford & Wu, 2018). Simple proteins oryzenin from rice, etc.
I. Background
1.4 Proteins 13
4. Prolamines: These are soluble in 70%80% 3. Chromoproteins: These are composed of
alcohol and insoluble in water, absolute simple proteins with chromotropic group as
alcohol and other neutral solvents. prosthetic group. Examples include
Examples are zein (corn), hordein (barley), hemoglobin (prosthetic group is heme),
gliadin (wheat), etc. flavoproteins (prosthetic group is
5. Albuminoids (Screlroproteins): Albuminoids riboflavin), cytochrome (prosthetic group is
are insoluble proteins and form supportive heme), etc.
tissues. These are animal proteins found in 4. Phosphoproteins: These are composed of
hair, nails, horns and hooves. Examples are proteins and phosphoric acid as the
keratin, collagen, gelatin, etc. prosthetic group. Caesin (milk protein) and
6. Histones: These are soluble in water, very vitelline (egg yolk protein) are the important
dilute acids and salt solutions. These are not examples of this group.
conjugated by heat and contain basic amino 5. Lipoproteins: These proteins are the
acids. Examples include nucleic acids. combination of proteins and lipids (fatty
7. Protamines: These are the simplest of acid, lecithin, cephalin, etc.) as prothetic
proteins and are basic polypeptides, soluble group. Lipoproteins occur in blood, cell
in water and ammonium hydroxides. These nuclei, milk, cell membranes, egg yolk, etc.
are not conjugated by heat. Examples 6. Metalloproteins: These are compounds of
include salmine (salmon sperm), clupeine proteins and some metals (such as iron,
(herring sperm), etc. cobalt, zinc, manganese, copper and
magnesium). Examples are ferritin,
ceruloplastin, carbonic anhydrase, etc.
1.4.2 Conjugated proteins
These contain simple protein combined with
1.4.3 Derived proteins
nonprotein prosthetic group (Murray et al.,
2006). These include: (1) nucleoproteins (2) pro- These are formed from simple and conjugate
teoglycans and glycoproteins (3) chromopro- proteins by denaturation or partial hydrolysis
teins (4) phosphoproteins (5) lipoproteins and (Murray et al., 2006). They are of two types (1)
(6) metalloproteins (Murray et al., 2006; denatured or primary derived proteins and (2)
Watford & Wu, 2018). secondary derived proteins (Murray et al.,
2006; Watford & Wu, 2018).
1. Nucleoproteins: These proteins are
composed of simple proteins with nucleic 1. Denatured or primary derived proteins:
acids as prosthetic group. In nucleoproteins, These proteins may be of different types.
protein moiety is usually a basic protein like a. Proteans—derived in the early stages of
protamine and histone. Examples include protein hydrolysis by water, dilute acids
chromosomal proteins and some glandular or alkalis or enzymes. Examples include
proteins. fibrin from fibrinogen, myosan from
2. Proteoglycans and glycoproteins: These myosin and edestan from edestin.
proteins contain carbohydrates as prosthetic b. Metaproteins—derived by further
group like hyaluronic acid and chondroitin hydrolysis by stronger acids or alkalies,
sulpahte. These are mainly found in blood which are insoluble in very dilute acids
plasma, gastric and salivary mucine, and alkalis. Examples of such proteins
immunoglobulins, human chorionic include acid metaproteins and alkali
gonadotropins, etc. metaproteins.
I. Background
14 1. Biological macromolecules: sources, properties, and functions
TABLE 1.3 Pharmacological effects of bioactive proteins/peptides along with their sources.
Bioactive proteins/
peptides Sources Pharmacological effects
Fibrinolytic Eisenia fetida Antitumor activity against several hepatoma cell lines, in vitro and in vivo (Chen
enzymes (earthworm) et al., 2007)
Hirudin Hirudo Anticoagulation activity through inhibition of thrombin activity (Markwardt, 2002)
medicinalis
Cordymin Cordyceps Antifungal activity (Wong et al., 2011), anticancer and inhibitory effect on HIV-1
militaris reverse transcriptase (Wong et al., 2019), antiinflammatory and antinociceptive
activities (Qian, Pan, & Guo, 2012)
Lectin Cordyceps Hemagglutinating activity and mitogenic activity (Wong, Wang, & Ng, 2009)
militaris
Ginkbilobin Ginkgo biloba Antifungal activity (Wang & Ng, 2000)
seeds
Dioscorin Dioscorea batatas Trypsin inhibitory activities (Hou et al., 1999)
Trichosanthin Trichosanthes Anti-HIV activity (Zhao, Ben, & Wu, 1999) and antiviral activity against hepatitis B
kirilowii virus (Wen et al., 2015)
I. Background
1.5 Nucleic acids 15
sugars (Minchin & Lodge, 2019; Nelson & Cox, diphosphate (ADP), guanosine triphosphate
2005). A nucleotide is composed of a pentose, a (GTP), guanosine diphosphate (GDP), cytidine
phosphate and a nitrogen base. The nitrogen triphosphate (CTP), cytidine diphosphate
base may be a purine or a pyrimidine. In case (CDP), uridine triphosphate (UTP), etc.
of RNA and DNA, the pentose is ribose and (Murray et al., 2006).
deoxyribose, respectively (Brosius & Raabe,
2016; Schwartz et al., 1991). Adenine and gua-
nine are the major purine bases (others are 1.5.2 Nucleosides
methyladenine, methylguanine, hypoxanthine,
It is a structural subunit of nucleic acids. In
etc.) whereas cytosine, uracil and thymine are
living cell, nucleoside is the heredity control-
the major pyrimidine bases (others include 5-
ling component (Murray et al., 2006; Nelson &
methylcytosine, 5, 6-dihydrouracil, etc.) of
Cox, 2005). When the ester bond between the
nucleic acids (Brosius & Raabe, 2016; Schwartz
sugar and the phosphate group in a nucleotide
et al., 1991). Various functions of nucleic acids
is hydrolyzed, a fragment consists of nitroge-
are (Minchin & Lodge, 2019; Nelson & Cox,
nous base and a sugar moiety is obtained
2005):
which is called as nucleoside. Sugar moiety in
1. Nucleic acids direct the metabolism process nucleoside is either ribose or deoxyribose,
of the cell throughout the life whereas nitrogenous bases consist of either a
2. Synthesis of protein is directed by nucleic pyridine, that is, cytosine, thymine, or uracil or
acids a purine, that is, adenine or guanine (Nelson &
3. They regulate the synthesis of enzymes Cox, 2005).
4. They play important role in the transfer of
genetic information from one offspring to
another 1.5.3 DNA
5. Nucleic acids contribute essential substances
DNA is a biological macromolecule where
of the genes and the apparatus by which the
genetic information of cell is confined, which is
genes act
known as genome of the cell (Nelson & Cox,
6. Nucleic acids are intimately involved with
2005; Watson & Crick, 1953). It is a polymer of
the varieties of disease like cancers, etc. and
deoxyribonucleotides. It occurs in chromo-
are major areas for research
somes, mitochondria and chloroplasts. The
chemical nature of monomeric units of DNA is
deoxyadenylate, deoxyguanylate, deoxycytidy-
1.5.1 Nucleotides late and thymidylate (Watson & Crick, 1953).
When the phosphate diester bond gets The monomeric units are held in polymeric
hydrolyzed, the monomeric nucleic acids are form by 30 , 50 -phophodiester bridges constitut-
separated which consist of nitrogenous base, a ing a single strand (Murray et al., 2006). The
sugar and a phosphate, and that unit is called genetic information resides in the sequence of
nucleotide (Nelson & Cox, 2005; Zaharevitz the monomeric unit. The polymer possesses a
et al., 1992). According to the presence of polarity, that is, one end has a 50 -hydroxyl or
ribose or deoxyribose, these may be ribonu- phosphate terminus while other has a 30 -phos-
cleotides or deoxyribonucleotides, respectively. phate or hydroxyl moiety. In DNA, the concen-
Nucleotides carrying more than one phosphate tration of adenosine nucleotide equals to
group are called higher nucleotides, for exam- thymidine and the concentration of guanosine
ple, adenosine triphosphate (ATP), adenosine nucleotide equals to cytosine nucleotide
I. Background
16 1. Biological macromolecules: sources, properties, and functions
(Nelson & Cox, 2005; Watson & Crick, 1953). 2. Cell replication—Hereditary characteristics
The secondary structure of DNA consists of a are passed on to daughter cells through
double stranded helix (Watson & Crick, 1953). replication of DNA.
The two strands of right handed DNA mole- 3. Control protein synthesis.
cules are held by hydrogen bonds. Each strand 4. Transcription and translation.
is again compactly held by hydrophobic forces
between the rings of its consecutive bases. The
pairing between the purine and pyrimidine 1.5.4 RNA
nucleotides on opposite strands is (Chang,
There are three types of RNA known to exist
2017) specific and dependent upon hydrogen
(Brosius & Raabe, 2016):
bonding of adenine (A) residue with thymine
(T) residue and guanine (G) with cytosine (C) 1. Messenger RNA (mRNA)
residue (Malhotra & Ali, 2018; Watson & Crick, 2. Transfer RNA (tRNA)
1953). Schematic representation of the structure 3. Ribosomal RNA (rRNA)
of DNA with its nitrogenous bases is presented
These are polymer of purine and pyrimidine
in Fig. 1.8 (right-hand side).
ribonucleotides linked together by phophodie-
Important biological roles of DNA are
ster bonds (Nelson & Cox, 2005). Schematic
(Nelson & Cox, 2005; Pisetsky, 2017; Watson &
representation of the structure of RNA with its
Crick, 1953):
nitrogenous bases is presented in Fig. 1.8 (left-
1. The function of DNA is to act as a storage hand side). Major nucleotides in RNA are
house of genetic information and to control adenylic, guanylic, cytidylic and uridylic acids.
the synthesis of protein in the cell. However, thymine is absent except in tRNA.
I. Background
1.5 Nucleic acids 17
RNA is distributed throughout the cell, most of anticodon group at the end of base paired
which remains present in cytoplasm as soluble stem recognizes the triplet nucleotide or
and rRNA, but about 10% is found in nucleus codon of the template mRNA. The DHU
with very small quantities being also present in loop helps to recognize the specific enzyme
the mitochondria (Higgs & Lehman, 2015; which activates the specific amino acids.
Nissen et al., 2000). The Thymidine-pseudouridine cytidine
binds the tRNA in ribosomes for protein
1. Messenger RNA (mRNA): These are
synthesis (Phizicky & Hopper, 2010).
homogenous in size and stability.
3. Ribosomal RNA (rRNA): It constitutes
Amongst all RNAs, mRNAs exhibit
nearly 50%60% of the total RNA of the cell
highest molecular weight (Sergeeva,
and is single stranded fibrous molecules
Koteliansky, & Zatsepin, 2016). An mRNA
which are highly elongated (Nelson & Cox,
carries adenine, guanine, cytosine and
2005; Urlaub, Kruft, Bischof, Müller, &
uracil as the major bases along with some
Wittmann-Liebold, 1995). An mRNA carries
minor bases, such as methylpurines and
adenine, guanine, cytosine and uracil as the
methylpyrimidines (Guan & Rosenecker,
major bases along with some minor bases
2017). mRNAs give signal for the
such as methylpurines and
synthesis of very important substances
methylpyrimidines. One or more segments
like the enzymes, the proteins, a variety
of mRNA strand carry the genetic code or
of polypeptide hormones, etc. (Guan
message, which is translated into the
& Rosenecker, 2017; Sergeeva
primary structure of a protein. Each genetic
et al., 2016).
code consists of many consecutive
2. Transfer RNA (tRNA): This consists of
nucleotide triplets called codons, each of
approximately 75 nucleotides and generated
which helps to incorporate specific amino
by nuclear processing of precursor molecule
acids in the peptide being synthesized
(Balatti, Pekarsky, & Croce, 2017; Phizicky &
(Higgs & Lehman, 2015).
Hopper, 2010). It serves as an adapter
molecule for the translation of information Nucleic Acids, that is, DNA and RNA are
in sequence of nucleotides of mRNA into significantly employed as biomedicine for the
specific amino acids. tRNA is participated in management of varieties of physiological con-
protein synthesis (Phizicky & Hopper, 2010). ditions (Minchin & Lodge, 2019). In a study, it
Beside the presence of regular bases, that is, was observed that chitosan nanoparticles
adenine, guanine, uracil and cytosine, tRNA loaded with probiotic DNA showed hypogly-
has been found to contain some very cemic activity (Kaur, Bhatia, Sethi, Kaur, &
unusual bases like ribothymidine, Vig, 2017). Antidiabetic activity has been
dihydrouracil, inosine, dihydrouridine reported by some other researchers by combin-
(DHU), pseudouridine, etc., which possess ing of berberine and noncoding RNA (Chang,
an unusual linkage in-between the sugar 2017). Floxuridine, a cytotoxic nucleoside ana-
ribose and the base (Higgs & Lehman, 2015; log, is a very good anticancer drug and it can
Nelson & Cox, 2005). All tRNA molecules be incorporated into DNA strands by synthesis
consist of an ACC sequence at the 30 termini. or incorporated into RNA by transcription (Ma
It is through an ester bond to the 30 - et al., 2018). This can be used as a real nucleo-
hydroxyl group of the adenosyl moiety that side. DNases II of tumor cells hydrolyze the
the carboxyl groups of amino acids are nucleotide strands and cytotoxic drug is
attached (Phizicky & Hopper, 2010). The released.
I. Background
18 1. Biological macromolecules: sources, properties, and functions
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posite scaffolds for cardiac tissue regeneration applica-
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I. Background
C H A P T E R
2
Structure activity relationship of
biological macromolecules
Aurelie Sarah Mok Tsze Chung, Yong Kiat Teo, Wai Teng Cheng
and Joash Ban Lee Tan
School of Science, Monash University Malaysia, Bandar Sunway, Malaysia
Biological Macromolecules
DOI: https://doi.org/10.1016/B978-0-323-85759-8.00002-6 23 © 2022 Elsevier Inc. All rights reserved.
24 2. Structure activity relationship of biological macromolecules
I. Background
2.2 Enzymes as bioactive proteins 25
biomedical field have been actively growing oxidative, proteolytic and quorum-quenching
over the years. While enzymes are essential for enzymes respectively (Sabala et al., 2012;
fundamental physiological functions, they also Sikdar & Elias, 2020; Ullah, 2020). The selection
possess several bioactive therapeutic proper- of these specific enzymes highlights the thera-
ties. Some enzymes can be used as antitumor peutic potential of enzymes, while also under-
and antimicrobial drugs, whereas others can be pinning their associated structural diversity.
used to treat genetic disorders and cardiovas-
cular diseases (Baldo, 2015; Gurung, Ray, Bose,
& Rai, 2013; Sabala, Jonsson, Tarkowski,
& Bochtler, 2012; Thallinger, Prasetyo,
2.2.1 L-amino acid oxidases
Nyanhongo, & Guebitz, 2013). Moreover, The LAOs are one of the most-studied mem-
unlike conventional drugs, enzymes are highly bers of the flavoenzyme family. They are
specific to their substrate, to which they can widely distributed in nature across diverse
bind and act on their targets with great affinity phyla, from fungi and bacteria to plants and
(Robinson, 2015). The spatial arrangement and animals (Sabotič et al., 2020; Yang et al., 2011).
type of amino acids at the active site can pro- In particular, LAOs from snake venoms (SV)
vide a conformation complementary to that of have been extensively investigated to probe
the substrate, allowing considerable specificity understanding on their ability to induce toxic
in their catalytic activity (Ghanem & Raushel, physiological effects and to develop snakebite
2012). In certain cases, some enzymes may also envenomation treatment (Hossain et al., 2014).
contain another component, known as a cofac- However, it was later demonstrated that SV-
tor, essential for their catalytic activity LAOs may additionally possess dose-
(Andreo-Vidal, Sanchez-Amat, & Campillo- dependent antibacterial, antiparasitic, antifun-
Brocal, 2018). Other than that, the rest of the gal and antitumor properties (Costa et al.,
enzyme structure stabilizes the active site, cre- 2015; Mukherjee et al., 2015; Rey-Suárez et al.,
ating a suitable environment for the interaction 2018; Soares et al., 2020; Zainal Abidin et al.,
of the site with the corresponding substrate 2018). These properties were also observed in
(Robinson, 2015). bacterial and fungal LAOs (Andreo-Vidal
In view of the vast array of enzymes with et al., 2018; Chen, Lin, Chen, Wang, & Sheu,
bioactive properties that exist, a few examples 2010; Yang et al., 2011). Hence, due to the high
of enzymes, whose crystalline structures have availability of LAOs in nature, their potential
been studied and deposited in the PDB have use for therapeutic purposes has been further
been selected for the discussion of their SAR. spurred.
These include the L-amino acid oxidases These enzymes catalyze the oxidative deam-
(LAOs), lysostaphin and metallo-β-lactamase- ination of L-amino acids with a strict stereo-
like lactonase (MLL). All three enzymes are specificity under aerobic conditions, producing
active against pathogenic bacteria, except for the corresponding α-ketoacids and ammonia,
LAO which is also active against tumorigenic while also generating hydrogen peroxide
cells (Cheleuitte-Nieves et al., 2020; Costa et al., (H2O2) (Wellner & Meister, 1961). They are
2015; López-Jácome et al., 2019; Mukherjee, composed of homodimers with a flavin ade-
Saviola, Burns, & Mackessy, 2015). Each of the nine dinucleotide (FAD) as a cofactor and each
aforementioned enzymes act through catalyz- protomer (50 70 kDa) contains three con-
ing a distinct type of reaction and using differ- served domains: the substrate-binding, FAD-
ent substrates. For these reasons, the LAO, binding and helical domains (Fig. 2.1)
lysostaphin and MLL are categorized as (Feliciano, Rustiguel, Soares, Sampaio, &
I. Background
26 2. Structure activity relationship of biological macromolecules
Cristina Nonato, 2017; Pawelek et al., 2000; which play a functional role for their antibacte-
Sabotič et al., 2020; Wiezel et al., 2019). Few rial and antitumor activities (Soares et al., 2020;
exceptions have been reported where LAOs Ullah, 2020).
can exist as monomers or tetramers, with the Although the substrate specificity of LAOs
latter being only biologically active in their may vary, most LAOs demonstrate a high
multimeric form (Andreo-Vidal et al., 2018; affinity for hydrophobic L-amino acids
Georgieva, Murakami, Perband, Arni, & Betzel, (Andreo-Vidal et al., 2018; Naumann et al.,
2011; Rey-Suárez et al., 2018). A highly con- 2011; Rey-Suárez et al., 2018; Sabotič et al.,
served motif β-α-β and a glutamic acid-rich 2020; Soares et al., 2020; Wiezel et al., 2019).
motif are observed in the N-terminal sequence Nevertheless, there are exceptions where
of LAOs, important for the FAD binding hydrophilic L-amino acids can be the best sub-
(Izidoro et al., 2014; Yu, Zhou, Qiao, & Qiu, strate for some LAOs (Ben et al., 2019;
2014). Moreover, 3% 4% of the molecular Nuutinen, Marttinen, Soliymani, Hilden, &
mass of most LAOs consist of carbohydrates, Timonen, 2012). The variation in LAO
FIGURE 2.1 (A) Cartoon representation of the LAO dimer from the Malayan Pit viper Calloselasma rhodostoma.
Opposite charges on the surface of each protomer stabilize the functional dimeric form of the protein. The substrate-
binding, FAD-binding and helical domains for each protomer are colored in green, red and blue respectively. The glycan
moiety is located at the surface of the protein while the cofactor, FAD in buried inside each protomer. (B) Close-up view of
the protomer. Residues Ile374 and Ile430 account for substrate preference of the LAO from Viper a. ammodytes to L-phenyl-
alanine. Residue His223 plays an important role in regulating substrate specificities. Source: (A) From the RCSB PDB (rcsb.
org) of PDB ID 1F8R (Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., et al. (2000). The Protein Data
Bank. Nucleic Acids Research, 28(1), 235 242; Burley, S.K., Bhikadiya, B., Bi, C., Bittrich, S., Chen, L., Crichlow, G.V., et al.
(2020). RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied
research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. Nucleic Acids
Research, 49(1), 437 451; Pawelek, P.D., Cheah, J., Coulombe, R., Macheroux, P., Ghisla, S., Vrielink, A. (2000). The structure of L-
amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. The EMBO Journal, 19(16),
4204 4215) (Berman et al., 2000; Burley et al., 2020).
I. Background
2.2 Enzymes as bioactive proteins 27
substrate specificities may be explained by the antimicrobial activity of LAOs is summarized
differences in amino acid composition and in Fig. 2.2.
sequence in the loops region, which in turn Apart from their antimicrobial activity,
influence the surface charge distribution, as LAOs can also exert a cytotoxic and antiproli-
well as the cavity volume and depth at the ferative effect on different tumorigenic cells
active site (Ullah, 2020). For example, the such as leukemia, lung cancer, gastric cancer,
hydrophobic amino acid residues Ile374 and prostate cancer, breast cancer and colon carci-
Ile430 at the substrate-binding site account for noma cells (Costa et al., 2015; Li Lee, Chung,
the preference of the LAO from Vipera ammo- Yee Fung, Kanthimathi, & Hong Tan, 2014;
dytes ammodytes to L-phenylalanine (Georgieva Naumann et al., 2011; Salama et al., 2018;
et al., 2011). Interestingly, the amino acid resi- Zainal Abidin et al., 2018). It was reported
due at position 223 in the active site has been that the glycan moiety at position 172 on the
reported to play an important role in regulat- protein surface mediates the interaction of
ing the substrate specificities of LAOs. It was SV-LAOs with the cell surface for a targeted
observed that when alanine or serine occupied local release of H2O2. This further enhances
this position, the LAO from Daboia venoms oxidative stress, which consequently leads to
had higher specificity towards L-arginine as the DNA damage and cell apoptosis (Bedoya-
substrate-binding cavity was further expanded Medina et al., 2019; Bregge-Silva et al., 2012;
and the steric repulsion towards this substrate Feliciano et al., 2017; Geyer et al., 2001;
was reduced (Chen, Wang, Huang, Huang, & Machado et al., 2018; Naumann et al., 2011).
Tsai, 2012). Hence, position 223 could be fur- Furthermore, the LAOs can induce proteolytic
ther exploited for drug design. enzyme release, which is essential in pro-
The ability of LAOs to inhibit bacterial and grammed cell death via two pathways: via
fungal pathogens has been associated with the their interaction with death receptors in the
exogenous production of H2O2 during the oxi- plasma membrane of tumorigenic cells, such
dative deamination of L-amino acids, as this as the Fas receptor; or through the activation
effect was inhibited by the presence of catalase of the mitochondria-mediated caspase path-
(Andreo-Vidal et al., 2018; Costa et al., 2015; way upon depolarization of the mitochon-
Sabotič et al., 2020). It was also suggested that drial membrane due to the accumulation of
the most hydrophobic sequences in the amphi- ROS (Bedoya-Medina et al., 2019; Mukherjee
pathic N-terminus of LAAOs are able to inter- et al., 2015; Pišlar, Sabotič, Šlenc, Brzin, &
act with the cell surface of bacteria such as Kos, 2016; Tan, Ler, Gunaratne, Bay, &
Staphylococcus aureus and Escherichia coli, subse- Ponnampalam, 2017; Tavares et al., 2016;
quently destabilizing the cell membrane integ- Zhang & Cui, 2007). In the absence of the gly-
rity, which further enhanced the antimicrobial can moiety, the catalytic activity of the LAO
effect (Abdelkafi-Koubaa et al., 2016; Costa is not affected, but the apoptotic activity is
et al., 2015; Yang et al., 2011). The cell mem- significantly reduced (Ande et al., 2006; Lu
brane permeabilization may result in an accu- et al., 2018; Ullah, 2020). This demonstrates
mulation of H2O2, or the production of other that the apoptotic effect is not only brought
reactive-oxygen species (ROS) intracellularly about by the production of H2O2, but also
due to an unusual metabolic cytosol environ- depends on the interaction of the glycan moi-
ment. Consequently, cellular damages such as ety, thus underpinning its importance in the
lipid peroxidation and DNA fragmentation structure of LAOs.
may occur, leading to bacterial growth inhibi- In summary, based on the general struc-
tion (Yang et al., 2011). An illustration of the tural framework of LAOs, it can be observed
I. Background
28 2. Structure activity relationship of biological macromolecules
FIGURE 2.2 (A) Representation of the catalytic activity of LAO, using L-amino acids as a substrate to release H2O2 as
one of its by-products. (B) Binding of the glycan moiety to the cell surface receptor helps to produce a localized high con-
centration of H2O2, which may accumulate intracellularly. (C) Interaction between the most hydrophobic sequences in the
N-terminus with cell surface may destabilize the cell membrane integrity. (D) Formation of endogenous H2O2 due to
unusual metabolic environment. (E) Accumulation of ROS may lead to DNA fragmentation. (F) ROS production may lead
to lipid peroxidation in the membrane layer.
I. Background
2.2 Enzymes as bioactive proteins 29
TABLE 2.1 Summary of the important structural components of LAO, lysostaphin and MLL associated with their
bioactivities.
L-amino oxidase
Bioactivity Antibacterial and antitumor
Substrate L-amino acids
Cofactor FAD
Domains Substrate-binding domain, FAD-binding domain, Helical domain
Active site Amino acid at position 223 can alter substrate specificity
N-terminal Most hydrophobic sequences interact with bacterial surface to disrupt membrane integrity
Glycan moiety Position 172 mediates interaction with tumor cell surface for localized production of H2O2
Lysostaphin
Bioactivity Antibacterial and antibiofilm
Substrate Pentaglycine cross-bridges
Cofactor Zn21
Metallo-β-lactamase-like lactonase
responsible for its catalytic activity, and the C- et al., 2014). The catalytic (CAT) domain at the
terminal cell-wall targeting (CWT) domain for active site consists of an antiparallel β-sheet
the binding to the peptidoglycan layer which anchors catalytic residues, grouped
(Fig. 2.3) (Baba & Schneewind, 1996; Sabala around a tightly bound central Zn21 cofactor,
I. Background
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Title: Metsolassa
Language: Finnish
Kirj.
Oskari Hynninen
SISÄLLYS:
Metson soitimella.
Nevalla.
Heija-Pekko.
Eläimiemme talvipuvuista.
Koiralleni.
Alleja ampumassa.
Mateita pyytämässä.
Miten kesäpäiväni viettäisin.
Syysmuistoja.
Talvinen metsä.
Merilintuja.
Luvattomalla ajalla.
Hylkeenhuudossa.
Metsolassa.
Vappuna.
METSON SOITIMELLA.