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Methods in
Molecular Biology 2558

Claudia Binda Editor

Monoamine
Oxidase
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Monoamine Oxidase

Methods and Protocols

Edited by

Claudia Binda
Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
Editor
Claudia Binda
Department of Biology and Biotechnology
University of Pavia
Pavia, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2642-9 ISBN 978-1-0716-2643-6 (eBook)
https://doi.org/10.1007/978-1-0716-2643-6
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
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Preface

This volume of the Methods in Molecular Biology series is specifically dedicated to mono-
amine oxidases (MAOs). Among the different enzymes that catalyse the oxidation of a
carbon-nitrogen bond in bioactive substrates, MAOs play a key role in the central nervous
system by deaminating aromatic neurotransmitters such as dopamine, serotonin, and adren-
aline. Moreover, xenobiotic compounds containing primary, secondary, or even tertiary
amino groups that may be introduced pharmacologically or with one’s diet may be sub-
strates for MAOs. Two isozymes, MAO A and MAO B, are expressed in mammals, which
share 70% sequence identity and are both FAD-dependent enzymes anchored to the outer
mitochondrial membrane through a C-terminal α-helix.
MAOs have been known for almost a century, given that the identification of an
oxidative deamination activity on tyramine was published in 1928. Since then, a huge
number of studies on MAOs have been reported yielding more than 24,000 results by
searching PubMed with the keyword “monoamine oxidase”. Other eukaryotic MAO
enzymes were discovered such as fungal MAO N that is about 25% identical to MAO
A/MAO B and is devoid of the membrane-binding domain, which makes this enzyme
ideal for biocatalytic industrial applications that involve an amine oxidation step. This book
will focus on methods used to study mammalian (mainly human) MAO A and MAO B that,
for their central role in neurotransmitter metabolism, are validated drug targets for neuro-
pathologies such as Parkinson’s and Alzheimer’s diseases and depression. In addition, the
clinical context involving MAO A and MAO B is expanding as these enzymes are widely
expressed not only in the brain but also in other organs such as heart, liver, placenta, and
platelets. Their enzymatic activity, which generates high levels of hydrogen peroxide as
secondary product, is supposed to contribute to the onset of ageing-related cardiovascular
diseases and certain types of solid tumours by increasing cellular oxidative stress.
Mammalian MAOs have been extensively studied, both biochemically within the specific
context of flavoenzymes (covering also purely mechanistic aspects) and pharmacologically in
the framework of their involvement in neurological diseases. This book aims at providing a
picture of the main methods to study mammalian MAOs, ranging from cell biology to
computational chemistry. A collection of 17 chapters have been written by scientists actively
involved in this field who contributed to developing most of the described methods. The
first two chapters are focused on methods used to obtain pure samples of MAO A and
MAO B, either from natural tissues (Chap. 1) or as recombinant proteins (Chap. 2). The
former was routinely used before the advent of molecular biology techniques and may be
still useful when native tissue-specific enzymes are required, the latter entailed the develop-
ment of a eukaryotic expression system in Pichia pastoris which was then extensively used
also for other membrane proteins. Chapters 3, 4, 5, 6, 7, and 8 concern assays and
techniques used to measure MAO enzymatic activity and perform inhibition studies.
Chapter 9 is focused on protocol to crystallize human MAO B, which so far has been the
most characterized form from a structural point of view. Chapters 10, 11, 12, 13, and 14
describe different methods to address cellular localization and function of MAOs, either in
cell lines or in animal models. The last chapters (Chaps. 15, 16, and 17) are dedicated to
computational methods applied to rational drug design approaches that are used to develop
new MAO inhibitors.

v
vi Preface

Some of the methods described in this book were elaborated many years ago and they
are still used, in some cases optimized with technological improvements. For this reason, it
may be difficult to learn and reproducibly apply these MAO-specific methods simply
following the experimental section of articles. I hope this book may be useful for scientists
interested in studying MAOs and other similar amine oxidase enzymes.

Pavia, Italy Claudia Binda

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Purification of MAO A and MAO B from Mammalian Tissue Sources . . . . . . . . . 1
Dale E. Edmondson
2 Purification of Recombinant Eukaryotic MAO A and MAO B
Utilizing the Pichia pastoris Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Dale E. Edmondson
3 The Peroxidase-Coupled Assay to Measure MAO Enzymatic Activity. . . . . . . . . . 23
Joana Reis and Claudia Binda
4 Measurement of MAO Enzymatic Activity by Spectrophotometric
Direct Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Joana Reis and Claudia Binda
5 Radiochemical Assay of Monoamine Oxidase Activity . . . . . . . . . . . . . . . . . . . . . . . 45
Andrew Holt
6 MAO Visible Spectroscopy for Ligand Interactions, Redox Chemistry,
and Kinetics of Irreversible Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Rona R. Ramsay
7 Conventional Receptor Radioligand Binding Techniques Applied
to the Study of Monoamine Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Andrew Holt
8 Assay of MAO Inhibition by Chromatographic Techniques
(HPLC/HPLC-MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Tomás Herraiz
9 Crystallization of Human Monoamine Oxidase B . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Claudia Binda, Dale E. Edmondson, and Andrea Mattevi
10 Detecting Monoamine Oxidase A and B Proteins: A Western Blotting
Protocol and Some Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Jennifer N. K. Nyarko, Ryan M. Heistad, Paul R. Pennington,
and Darrell D. Mousseau
11 An (Immuno) Fluorescence Protocol for Monitoring Monoamine
Oxidase A/B Protein Distribution Within the Cell. . . . . . . . . . . . . . . . . . . . . . . . . . 143
Tyler J. Wenzel, Jennifer N. K. Nyarko, Ryan M. Heistad,
Paul R. Pennington, Chris P. Phenix, and Darrell D. Mousseau
12 Expression and Function of MAO A in Cardiac Cells by Means
of Adenovirus-Mediated Gene Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Yohan Santin, Angelo Parini, and Jeanne Mialet-Perez
13 In Vitro and In Vivo Assays Characterizing MAO A Function in Cancers . . . . . . 171
Boyang Jason Wu and Jean C. Shih

vii
viii Contents

14 In Vivo Study of Monoamine Oxidases Using Multisite Intracerebral

Microdialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Philippe De Deurwaerdère, Nouhad Samb, Hasna El Boukhari,
Rémi Corne, Abdeslam Chagraoui, and Giuseppe Di Giovanni
15 Informed Use of 3D-QSAR for the Rational Design of Coumarin
Derivatives as Potent and Selective MAO B Inhibitors . . . . . . . . . . . . . . . . . . . . . . . 197
Nicola Gambacorta, Marco Catto, Leonardo Pisani,
Angelo Carotti, and Orazio Nicolotti
16 Hansch-Type QSAR Models for the Rational Design of MAO
Inhibitors: Basic Principles and Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Leonardo Pisani, Modesto de Candia, Mariagrazia Rullo,
and Cosimo D. Altomare
17 Computational Chemistry and Molecular Modeling of Reversible
MAO Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Kemal Yelekçi and Safiye Sağ Erdem

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Contributors

COSIMO D. ALTOMARE • Department of Pharmacy-Pharmaceutical Sciences, University of

Bari “Aldo Moro”, Bari, Italy
CLAUDIA BINDA • Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
ANGELO CAROTTI • Department of Pharmacy-Pharmaceutical Sciences, University of Bari
“Aldo Moro”, Bari, Italy
MARCO CATTO • Department of Pharmacy-Pharmaceutical Sciences, University of Bari
“Aldo Moro”, Bari, Italy
ABDESLAM CHAGRAOUI • Normandie University, UNIROUEN, INSERM, U1239, CHU
Rouen, Neuronal and Neuroendocrine Differentiation and Communication Laboratory,
Institute for Research and Innovation in Biomedicine of Normandy (IRIB), Rouen,
France; Department of Medical Biochemistry, Rouen University Hospital, Rouen, France
RÉMI CORNE • Centre National de la Recherche Scientifique (Unité Mixte de Recherche
5287), Bordeaux Cedex, France
MODESTO DE CANDIA • Department of Pharmacy-Pharmaceutical Sciences, University of
Bari “Aldo Moro”, Bari, Italy
PHILIPPE DE DEURWAERDÈRE • Centre National de la Recherche Scientifique (Unité Mixte de
Recherche 5287), Bordeaux Cedex, France
GIUSEPPE DI GIOVANNI • Department of Physiology and Biochemistry, Faculty of Medicine
and Surgery, University of Malta, Msida, Malta; School of Biosciences, Cardiff University,
Cardiff, UK
DALE E. EDMONDSON • Department of Biochemistry, Emory University, Atlanta, GA, USA
HASNA EL BOUKHARI • Centre National de la Recherche Scientifique (Unité Mixte de
Recherche 5287), Bordeaux Cedex, France
SAFIYE SAĞ ERDEM • Department of Chemistry, Faculty of Arts and Sciences, Marmara
University, Istanbul, Turkey
NICOLA GAMBACORTA • Department of Pharmacy-Pharmaceutical Sciences, University of
Bari “Aldo Moro”, Bari, Italy
RYAN M. HEISTAD • Department of Psychiatry, University of Saskatchewan, Saskatoon, SK,
Canada
TOMÁS HERRAIZ • Instituto de Ciencia y Tecnologı́a de Alimentos y Nutricion (ICTAN),
Spanish National Research Council (CSIC), Madrid, Spain
ANDREW HOLT • Neurochemical Research Unit, Department of Psychiatry, Faculty of
Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
ANDREA MATTEVI • Department of Biology and Biotechnology, University of Pavia, Pavia,
Italy
JEANNE MIALET-PEREZ • Institute of Metabolic and Cardiovascular Diseases (I2MC),
INSERM, Université de Toulouse, Toulouse, France
DARRELL D. MOUSSEAU • Department of Psychiatry, University of Saskatchewan, Saskatoon,
SK, Canada
ORAZIO NICOLOTTI • Department of Pharmacy-Pharmaceutical Sciences, University of Bari
“Aldo Moro”, Bari, Italy
JENNIFER N. K. NYARKO • Department of Psychiatry, University of Saskatchewan, Saskatoon,
SK, Canada

ix
x Contributors

ANGELO PARINI • Institute of Metabolic and Cardiovascular Diseases (I2MC), INSERM,

Université de Toulouse, Toulouse, France
PAUL R. PENNINGTON • Department of Psychiatry, University of Saskatchewan, Saskatoon,
SK, Canada
CHRIS P. PHENIX • Department of Chemistry, University of Saskatchewan, Saskatoon, SK,
Canada
LEONARDO PISANI • Department of Pharmacy-Pharmaceutical Sciences, University of Bari
“Aldo Moro”, Bari, Italy
RONA R. RAMSAY • School of Biology, Biomolecular Sciences Research Complex, University of
St Andrews, Fife, UK
JOANA REIS • Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
MARIAGRAZIA RULLO • Department of Pharmacy-Pharmaceutical Sciences, University of
Bari “Aldo Moro”, Bari, Italy
NOUHAD SAMB • Centre National de la Recherche Scientifique (Unité Mixte de Recherche
5287), Bordeaux Cedex, France
YOHAN SANTIN • Institute of Metabolic and Cardiovascular Diseases (I2MC), INSERM,
Université de Toulouse, Toulouse, France
JEAN C. SHIH • Department of Pharmacology and Pharmaceutical Sciences, School of
Pharmacy, University of Southern California, Los Angeles, CA, USA
TYLER J. WENZEL • Department of Psychiatry, University of Saskatchewan, Saskatoon, SK,
Canada
BOYANG JASON WU • Department of Pharmaceutical Sciences, College of Pharmacy and
Pharmaceutical Sciences, Washington State University, Spokane, WA, USA
KEMAL YELEKÇI • Department of Bioinformatics and Genetics, Faculty of Engineering and
Natural Sciences, Kadir Has University, Istanbul, Turkey
 Chapter 1

Purification of MAO A and MAO B from Mammalian Tissue

Sources
Dale E. Edmondson

Abstract
Procedures are described for the purification of the mitochondrial-bound enzymes human and bovine
monoamine oxidases A and B (MAO A and B) from placental and liver tissue sources, respectively. Enzyme
purification follows isolation of the mitochondria and preparation of outer membrane particles. The
membrane-bound enzymes are solubilized by treatment of membranes with phospholipases and detergent
extraction. Functional bovine MAO B is purified by polymer fractionation and differential centrifugation.
Functional human MAO A is purified by ion-exchange DEAE-Sepharose chromatography.

Key words Placental MAO A, Bovine liver MAO B, Triton X-100 solubilization, Detergent, Ion-
exchange chromatography

1 Introduction

Monoamine oxidases A and B (MAOs) are mitochondrial outer-

membrane-localized enzymes, which are present in mammalian
tissues as two separate gene products [1, 2]. MAO A and MAO B
share ~70% identity and exhibit similar properties which make their
isolation and purification from tissues expressing both enzymes a
difficult if not impossible task, especially for biochemical studies
requiring reagent quantities of the target proteins. Typically, beef
liver mitochondria are a rich source of MAO B which can be
purified without contamination by MAO A [3]. MAO A has been
purified from human placental mitochondria [4] in quantities suit-
able for biochemical studies without MAO B contamination.
Therefore, prior to the development of heterologous expression
systems and purification procedures outlined in Chap. 2, human
placental tissue served as a source of MAO A and bovine liver as a
source of MAO B.
The procedures described below are based on the work of
Dr. James I. Salach in collaboration with Dr. Walter Weyler

Claudia Binda (ed.), Monoamine Oxidase: Methods and Protocols, Methods in Molecular Biology, vol. 2558,
https://doi.org/10.1007/978-1-0716-2643-6_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

1
2 Dale E. Edmondson

[5]. A very detailed description with full updates of the initial

published procedures was published approximately 25 years ago
[6]. The author of this chapter followed these procedures and
found that they can be reproduced readily in another laboratory;
therefore, he wishes to dedicate this chapter in memory of
Dr. Salach.

2 Materials

The approaches used to purify the membrane-bound MAOs from

mammalian tissues include initially the preparation of mitochondria
where MAO is localized and a further fractionation step to prepare
mitochondrial outer membranes from human placental tissue for
MAO A and bovine liver for MAO B. Among the solutions and
reagents listed in the following sections, some are used for both
MAO A and MAO B purification, whereas others are specific for the
protocol of one isozyme (and are not necessary for the other): it is
suggested to read the protocol to be applied to have the specific list
of materials to be prepared. All buffers are prepared using glass
distilled water and, unless otherwise stated, stored and used at 4
C.

2.1 Mammalian 1. Human placenta is obtained from a local hospital (see Note 1).
Tissues 2. Fresh bovine liver (4–6 kg) is obtained from a local
slaughterhouse.

2.2 Solutions Used 1. 0.25 M sucrose in 10 mM potassium phosphate, pH 7.0.

in the Preparation of 2. 0.25 M sucrose in 10 mM potassium phosphate, 0.5 mM
Human Placental and EDTA, pH 7.0, at 4
C.
Bovine Liver
3. 0.1 M KCl in 10 mM Tris/H3PO4, pH 7.2, at 4
C.
Mitochondria
4. 0.1 M KCl in 10 mM Tris/H3PO4, 0.5 mM EDTA, pH 7.2, at
4
C.
5. 50 mM phenylmethylsulfonyl fluoride (PMSF) in absolute
ethanol.

2.3 Buffers and 1. TEA buffer: 0.1 M triethanolamine/HCl, pH 7.2.

Reagents Used for 2. TEA buffer, pH 8: 50 mM triethanolamine/HCl, pH 8.0.
Purification of Human
3. Calcium chloride solution: 1 M CaCl2.
Placental MAO A and
Bovine Liver MAO B 4. Biuret reagent to measure protein concentration.
5. Phospholipase A and phospholipase C.
6. Ammonia solution: 2 M NH4OH.
7. Dextran (Mav ¼ 500.000) powder.
8. Polyethylene glycol (PEG) (Mav ¼ 8.000) powder.
9. Ficoll (Mav ¼ 400,000) powder.
 MAO Purification from Mammalian Tissue Sources 3

10. Triton solution: 20% w/v Triton X-100.

11. DEAE buffer A: 20 mM sodium phosphate, pH 7.2, contain-
ing 20% (w/v) glycerol.
12. DEAE buffer B: 250 mM sodium phosphate, pH 7.2, contain-
ing 20% (w/v) glycerol.
13. MAO B buffer: 50 mM sodium phosphate, pH 7.2, 50% (w/v)
glycerol.
14. Mercaptoethanol.
15. Octylglucoside powder.
16. d-Amphetamine powder.

2.4 Instruments and 1. Meat grinder.

Appliances 2. Waring blender equipped with Polytron blades.
3. High-speed centrifuge (up to 150,000 g).
4. Swinging bucket centrifuge (up to 10,000 g).
5. Handheld homogenizer (Fig. 1a) or motor-driven glass/Tef-
lon homogenizer (Fig. 1b).
6. Cheesecloth.
7. Magnetic stirrer.
8. 2  87 cm column of DEAE-Sepharose CL-6B.
9. Amicon Ultra 30 K centrifugal filter (Millipore).

Fig. 1 Tools to homogenize pellet after high-speed centrifugation. (a) Handheld homogenizer consisting of a
glass tube and a Teflon pestle that perfectly fits the tube for an optimal homogenization. (b) Motor-driven
machine to mount the glass/Teflon homogenizer shown in (a) that is useful to speed-up the process
4 Dale E. Edmondson

10. 18-gauge syringe with needle.

11. UV/Vis spectrophotometer (to determine protein concentra-
tion by biuret assay and to detect protein during purification).
12. FPLC system (although chromatographic runs can be also
carried out by gravity).

3 Methods

Unless otherwise stated, all procedures must be performed at 4
C.

3.1 Preparation of 1. Place human placenta obtained from hospital on ice immedi-
Human Placental ately after delivery and plan to start mitochondrial preparation
Mitochondria on return to the laboratory. Remove the umbilical cord and
amniotic sac and cut the remaining tissue into 1-cm-sized
pieces.
2. Pass the tissue through a meat grinder and immediately resus-
pend it in 1.5 L of solution 2 containing 15 mL of solution
5 resulting in 0.5 mM PMSF. The suspension is then homo-
genized for 45 sec using a Waring blender equipped with
Polytron blades and operated at a voltage (using a variable
transformer) of 55–60 V.
3. Centrifuge the homogenate at 1250 g for 15 min. The super-
natant is collected and centrifuged at 26,000 g for 15 min. The
resulting pellet (containing the mitochondrial fraction) is
washed by resuspending in 500 mL of solution 2 containing
0.5 mM PMSF using a handheld homogenizer and centrifuged
as stated above. The mitochondria are washed again by homog-
enization of the pellet in 70 mL of solution 4 and collected by
centrifugation at 40,000 g for 20 min. The pellet contains
25 units of MAO A activity (see Note 2) and can be stored
for at least 6 months at 70
C.

3.2 Preparation of 1. Place fresh bovine liver (4–6 kg) from a local slaughterhouse on
Bovine Liver ice. On return to the laboratory, the liver is freed of connective
Mitochondria tissue, cut into inch cubes, and washed with three changes of
solution 3 to remove most of the blood. The washed tissue is
then passed through a meat grinder into 4 L of solution 2.
2. Stir and homogenize the tissue suspension by a motor-driven
loose fitting glass/Teflon homogenizer using two vertical
passes. The suspension is diluted to 7 L with solution 2 and
centrifuged at 400 g for 15 min to remove unbroken cells,
nuclei, and other cellular debris. The supernatant is decanted
through cheesecloth and centrifuged at 10,400 g for 15 min to
sediment the mitochondrial fraction. The pellet is homoge-
nized with a minimal volume of solution 1 (taking care not to
 MAO Purification from Mammalian Tissue Sources 5

disturb the small red pellet due to any contaminating red blood
cells), diluted to 1.4 L with solution 1, and centrifuged at
7500 g for 15 min.
3. Resuspend and homogenize the washed pellet with solution
3, dilute to 500–600 mL, and centrifuge again at 7500 g for
15 min. The washed mitochondrial pellets exhibit
500–600 units of MAO B activity (see Note 2) and can be
stored at 20
C for several months without loss of activity.
The mitochondrial pellets are best stored in 50–100 mL
volumes.

3.3 Extraction and 1. Resuspend mitochondria pooled from 6 to 7 placenta

Purification of Human (140–150 units of catalytic activity) in TEA buffer and homog-
Placental MAO A enize using a hand homogenizer. The protein concentration is
measured with the biuret assay and adjusted to 20 mg/mL
(25
C).
2. Add calcium chloride solution added to a final concentration of
25 mM, followed by 670 units of phospholipase A and 1 mg of
phospholipase C (see Note 3) per 500 mg of mitochondrial
protein. The digestion is carried out with stirring of 1 h main-
taining the pH at 7.3 by the addition of aliquots of ammonia
solution. The digest is centrifuged at 45,000 g for 15 min at
15
C.
3. Wash the pellet by homogenization in TEA buffer and centri-
fugation is repeated. The resulting washed pellet is homoge-
nized in TEA buffer to a protein concentration of 15 mg/mL
and protein extraction is performed by the addition of Triton
solution to a stoichiometry of 1 mg detergent per 3 mg of
protein and the mixture is stirred for 30 min at 25
C. The
suspension is centrifuged at 45,000 g for 15 min at 15
C and
the supernatant containing the solubilized enzyme is recentri-
fuged to minimize turbidity.
4. Polymer fractionation of solubilized MAO A is achieved by the
addition of dextran (Mav ¼ 500.000) 0.5 g and polyethylene
glycol (PEG) (Mav ¼ 8.000), 0.4 g for each 8 mL of Triton
extract. The solids are dissolved and the mixture is stirred for
30 min at 20
C. The solution is centrifuged in a swinging
bucket rotor at 9500 g, 20
C, for 20 min. The rotor is allowed
to stop without braking resulting in a two-phase separation in
which MAO A is distributed in the upper phase. The yellow
phase containing MAO A is withdrawn with a syringe and
pooled, and PEG is added to a final concentration of 25%
(w/v). The mixture is stirred for 30 min at 20
C and the
precipitated protein is removed by centrifugation (17,000 g,
4
C, 10 min). The pellet is suspended by homogenization in a
loose-fitting glass/Teflon homogenizer in 8 mL of DEAE
buffer A and stored overnight at 4
C.
6 Dale E. Edmondson

5. Equilibrate the 2  87 cm column of degassed DEAE-

Sepharose CL-6B with 1 L of DEAE buffer A containing
3 mM mercaptoethanol (added upon usage). Octylglucoside
(0.8% w/v) and 1 mM d-amphetamine (final concentrations)
are added to the last 90 mL of equilibration buffer (see Note 4).
6. Dilute the MAO A solution obtained from step 4 to a protein
concentration of 13 mg/mL with DEAE buffer A and centri-
fuge at 150,000 g for 1 h at 4
C. The supernatant solution is
then incubated for 5 min at 25
C with 2 mg of octylglucoside
per mg of protein.
7. Apply this solution to the column and start elution with a linear
gradient of 20–250 mM with a total volume of 1220 mL
combining degassed DEAE buffer A and DEAE buffer B
accordingly (both solutions contain 3 mM mercaptoethanol,
0.8% octylglucoside, and 1 mM d-amphetamine added upon
usage). Fractions of 6 mL are collected with a flow rate of
36 mL/h MAO A containing fractions are monitored by activ-
ity measurements and, by absorption spectral measurements,
pooled and concentrated by ultrafiltration using an Amicon
Ultra 30 K centrifugal filter. Glycerol is added to the concen-
trated enzyme solution to 50% (w/v) and the enzyme stored at
20
C until use (see Note 5).

3.4 Preparation of 1. Thaw a slurry (100 mL) of bovine liver mitochondria overnight
Mitochondrial Outer at 5
C (see Note 6). The thawed mitochondrial slurry is
Membranes and homogenized into five volumes of cold distilled water using a
Purification of Bovine tight-fitting glass/Teflon homogenizer. The homogenate is
Liver MAO B further diluted with cold distilled water to tenfold increase in
volume over the initial volume of mitochondria. The mixture is
incubated for 15 min at 5
C to achieve osmotic shock of the
mitochondria to allow outer membrane preparation.
2. Centrifuge the mixture at 100000 g for 15 min at 5
C. The
supernatant is discarded and the pellet homogenized into TEA
buffer at room temperature. The homogenate is diluted with
fresh TEA buffer to a protein concentration of 20–30 mg/mL.
Calcium chloride solution is added to a final concentration of
25 mM.
3. Add 1 mg of phospholipase A/300 mg of protein and 1 mg of
phospholipase C/500 mg of protein (see Note 3) to the
homogenate and the mixture is incubated at 30
C with gentle
stirring. The pH is adjusted to 7.2 with aliquots of ammonia
solution and maintained as necessary for the 2-h incubation.
Most of the pH changes occur during the first hour. The digest
is then centrifuged at 100,000 g for 15 min at 20
C. The
pellets are combined and stored under fresh TEA buffer at 5
C
in the dark overnight.
 MAO Purification from Mammalian Tissue Sources 7

4. The next day, homogenize the pellets in fresh TEA buffer at

room temperature and adjust to a final concentration of
10–15 mg/mL. Triton solution is added to the sample to
achieve a 1:3 (w/w) ratio of detergent to total protein. The
sample is homogenized with a glass/Teflon homogenizer and
stirred for 30 min at room temperature. After centrifugation at
100,000 g for 15 min at 20
C, the supernatant is retained and
the pellet homogenized with one-fourth of the initial volume
of the extract and centrifuged at 100,000 g as above. The
supernatants are combined and the pellet discarded. The Triton
X-100 concentration of the pooled supernatant is adjusted to a
final concentration of 3 mg/mL.
5. For each 4 mL of extract, the following polymers are weighed
out – dextran (Mav ¼ 250,000) (0.44 g), Ficoll (Mav ¼ 400,000)
(0.48 g), polyethylene glycol 8000 (0.32 g) – and mixed
together in a 2-L beaker, and then 0.76 mL distilled water
and Triton extract are added. The mixture is stirred at room
temperature with an overhead stirrer until all of the solids are
dissolved. The emulsion is poured into centrifuge tubes and let
stand about 20–30 min to allow equilibration.
6. Centrifuge the solution in a swinging bucket rotor at 10000 g
at 20
C for 15 min. The centrifuge is allowed to stop with the
brake off. MAO B is located in the compact interface. Carefully
remove the upper and lower layers by aspiration and homoge-
nize the interfacial layer in cold TEA buffer, adjust the protein
concentration to 10 mg/mL, and centrifuge for 20 min at
41000 g at 4
C. The supernatant is decanted and may be
stored overnight at 0
C.
7. Centrifuge the supernatant (clear yellow enzyme solution) at
252000 g for 90 min (Beckman Ti60 rotor at 50000 rpm).
Remove the supernatant and homogenize the yellow pellet into
a minimal volume several mL of the MAO B buffer and stored
at 20
C. At this point in the purification, the enzyme is about
80% pure at this step and contains some heme impurity. Final
purification is achieved by sucrose density centrifugation
detailed in step 8 (see also Note 7).
8. Dilute the enzyme obtained from the above steps to a protein
concentration of 6 mg/mL with TEA buffer at pH 8. Discon-
tinuous sucrose density gradients are formed in the following
order: one part 60% (w/v) sucrose, one part 55% sucrose, one
part 50% sucrose, two parts 45% (w/v) sucrose, and one part
35% (w/v) sucrose (all sucrose solutions prepared in TEA
buffer at pH 8). Gradients are formed by layering 5 mL of
each sucrose solution into centrifuge tubes followed by the
enzyme solution layered at the top of the gradient. The gradi-
ents are centrifuged in a swinging bucket rotor (with the brake
8 Dale E. Edmondson

off) at 107,000 g at 4
C for 17 h. The developed gradients

contain two broad yellow bands containing MAO B activity
below a pink-red heme protein band below a white turbid
band. The MAO B bands are harvested by puncturing the
sides of the centrifuge tubes with an 18-gauge syringe needle.
9. Dilute the MAO B fraction approximately fourfold with
50 mM sodium phosphate, pH 7.2, at 5
C and centrifuge at
252,000 g for 90 min at 5
C. The resulting yellow pellet is
homogenized into a minimal volume of MAO B buffer and
stored at 20
C in the dark. The enzyme is stable under these
conditions for at least 6 months. If the enzyme is to be used
immediately, the pellet can be homogenized into MAO B
buffer and stored on ice in the dark for at least a month. The
specific activity of the purified enzyme is 4.5 units/mg which is
a 166-fold purification over the specific activity of and a 37%
yield of activity observed in the mitochondrial fraction.

4 Notes

1. Human placentas are obtained from a local hospital from

healthy volunteers. All patients signed permission forms and
all procedures were conducted in accordance with Institutional
Review Board regulations.
2. For assays used to measure MAO activity, the reader can refer
to Chaps. 3, 4, and 5 of this book. During MAO purification,
convenient assay protocols are briefly described as follows.
MAO A catalytic activity: Kynuramine (1 mM) oxidase activity
[7] is measured spectrophotometrically at 316 nm (Δε
¼12,000 M1cm1) at 25
C in 50 mM potassium phos-
phate buffer, pH 7.5, containing 0.5% (w/v) reduced Triton
X-100 at air saturation. MAO B catalytic activity: Benzylamine
(3 mM) oxidase activity [8] is also measured spectrophotomet-
rically at 250 nm at 25
C (Δε ¼12,800 M1cm1) in 50 mM
potassium phosphate buffer, pH 7.5, containing 0.5% (w/v)
reduced Triton X-100 at air saturation. It should be noted that
the [O2] at air saturation is saturating for MAO A and is close
to the Km(O2) for MAO B.
3. Phospholipase A is partially purified from Naja naja siamensis
lyophilized venom (Latoxan Laboratory, Valence, France) by a
modification of a published procedure [9]. After treatment at
100
C, pH 3.0, the venom solution is cooled and centrifuged
at 150000 g for 1 h. The supernatant is adjusted to pH 7.5 with
3 M NH4OH, centrifuged as before, and chromatographed on
a column of Bio-Gel P-50 equilibrated with 50 mM Tris-HCl,
pH 7.6. Enzyme-containing fractions are assayed by the pH
 MAO Purification from Mammalian Tissue Sources 9

changes on lecithin hydrolysis [10]. One unit of enzyme is

defined as the amount that liberates 1 μmole of protons/min.
Phospholipase C is obtained from Cl. welchii (Sigma-Aldrich).
4. d-Amphetamine is a competitive inhibitor of MAO A
(Ki ¼ 14 μM) and is routinely used to stabilize the enzyme
on purification and storage. It should be removed before con-
ducting any kinetic studies. The Drug Enforcement Adminis-
tration in the USA has classified this compound as a controlled
Schedule II drug and requires a license for research uses and
requires the compound to be stored in a locked drawer with
access only by qualified personnel.
5. For experimental use, glycerol and d-amphetamine
(a competitive inhibitor of MAO A) are removed by dialysis
or by gel filtration. The specific activity of the MAO A prepara-
tion is 1.7 units/mg with a 35% yield in activity relative to the
activity exhibited in the mitochondrial fraction with a 100-fold
increase in specific activity.
6. If any plasticware is to be used during the MAO B purifications,
the reader is strongly encouraged to wash the vessels with
methanol to remove contaminants found on plastic labware
which are found to be potent MAO B inhibitors [11].
7. It is possible that bovine MAO B can be further purified using
column chromatography on Q-Sepharose as is used with
human MAO B [12]. In our original publication on the expres-
sion and purification of human MAO B in Pichia [13], the
polymer fractionation procedure used for bovine MAO B was
successfully used. Therefore, it seems reasonable that the col-
umn fraction should also work with the bovine enzyme.

Acknowledgments

This work was funded by a grant from the National Institutes of

General Medical Sciences (GM-29433). The following coworkers
provided valuable contributions and include Milagros Aldecco,
Paige Newton-Vinson, and Mark Walker.

References
1. Shih JC (2018) Monoamine oxidase isoen-
zymes: genes, functions and targets for behav-
ior and cancer therapy. J Neural Transm
(Vienna) 125:1553–1566
2. Edmondson DE, Binda C (2018) Monoamine
Oxidases. Subcell Biochem 87:117–139
3. Salach JI (1979) Monoamine oxidase from
beef liver mitochondria: simplified isolation
procedure, properties, and determination of
its Cysteinyl Flavin content. Arch of Biochem
and Biophys 192:128–137
4. Weyler W, Salach JI (1985) Purification and
properties of mitochondrial monoamine oxi-
dase a from human placenta. J Biol Chem
260:13199–13207
5. Salach JI, Weyler W (1981) Iron content and
spectral properties of highly purified bovine
10 Dale E. Edmondson
liver monoamine oxidase. Arch Biochem Bio-
phys 212:147–153
6. Salach JI, Weyler W (1987) Preparation and
properties of the Flavin-containing aromatic
amine oxidases of human placenta and beef
liver. Meth Enzymol 142:627–637
7. Weissbach H, Smith TE, Witkop B, Uden-
friend S (1960) A rapid spectrophotometric
assay of monoamine oxidase based on the rate
of disappearance by Kynuramine. J Biol Chem
235:1160–1163
8. Tabor CW, Tabor H, Rosenthal SM (1954)
Purification of an amine oxidase from beef
plasma. J Biol Chem 208:645–661
9. Cremona T, Kearney EB (1964) Studies of the
respiratory chain-linked nicotinamide dehy-
drogenase VI further purification and proper-
ties of the enzyme from beaf heart. J Biol Chem
239:2328–2334
10. Salach JI, Seng R, Tisdale H, Singer TP (1971)
Phospholipases of Snake venoms II. Catalytic
properties of the enzyme from naja naja. J Biol
Chem 246:340–347
11. McDonald GR, Hudson AL, Dunn SMJ,
You H, Baker GB, Whittel RM, Martin JW,
Jha A, Edmondson DE, Holt A (2008) Bioac-
tive contaminants leach from disposable labo-
ratory plasticware. Science 322:917
12. Hubálek F, Binda C, Khalil A, Li M, Mattevi A,
Castagnoli N, Edmondson DE (2005) Dem-
onstration of isoleucine 199 as a structural
determinant for the selective inhibition of
human monoamine oxidase B by reversible
inhibitors. J Biol Chem 280:15761–15766
13. Newton-Vinson P, Hubalek F, Edmondson
DE (2000) High-level expression of human
liver monoamine oxidase B in Pichia pastoris.
Protein Expr Purif 20:334–345
 Chapter 2

Purification of Recombinant Eukaryotic MAO A and MAO B

Utilizing the Pichia pastoris Expression System
Dale E. Edmondson

Abstract
Procedures are described for the heterologous expression and purification of the mitochondrial-bound
enzymes human and rat monoamine oxidases A and B and zebrafish MAO in the yeast Pichia pastoris.
Enzyme expression is under control of a methanol oxidase promoter and similar procedures have been
developed for the preparation of membrane particles and detergent solubilization of the functional
enzymes. Similarities and differences are described in the procedures for purification of the respective
enzymes using standard column chromatographic techniques to provide enzyme yields in the range of
100–300 mg from 1 L of cell culture.

Key words Pichia pastoris, Recombinant Monoamine Oxidase, Triton X-100 solubilization,
Ion-exchange chromatography, Ceramic hydroxyapatite chromatography

1 Introduction

Monoamine oxidases (MAOs) are FAD-dependent enzymes bound

to the outer membrane of the mitochondria, which in mammals are
present as two separate gene products, namely, MAO A and MAO
B (for comprehensive reviews on this topic, see the introductive
section of Chap. 1). Teleosts differ from mammals in that only a
single gene encoding MAO is present and thought to be an evolu-
tionary precursor to a gene duplication event encoding separate
MAOs in mammals which exhibit ~70% identity. Due to their
membrane association and similar sizes and properties, the isolation
of MAO A and MAO B in quantities suitable for biochemical
studies has been a difficult task. Typically, beef liver mitochondria
are a rich source of MAO B which can be purified without contam-
ination by MAO A [1]. MAO A has been purified from human
placental mitochondria [2] in quantities suitable for biochemical
studies without MAO B contamination. Detailed procedures for
these tasks can be found in Chap. 1 of this book.

Claudia Binda (ed.), Monoamine Oxidase: Methods and Protocols, Methods in Molecular Biology, vol. 2558,
https://doi.org/10.1007/978-1-0716-2643-6_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

11
12 Dale E. Edmondson

There are a number of advantages to support the development

of a high-level expression system to produce recombinant active
MAOs. The first is that the procurement of fresh bovine and human
tissues is becoming increasingly more difficult. Given the increasing
importance of MAO A and MAO B in drug development by the
pharmaceutical industry, it is important to express both human and
rat MAO A and MAO B since rats are used in animal drug studies as
models for drugs that are eventually used in humans. A detailed
comparative study of human and rat MAO A and MAO B would
provide insights into their relative activities and affinities for com-
pounds under investigation. Zebrafish studies have become impor-
tant as a model animal system for neurochemical investigations
[3]. Since only a single MAO gene is found in this organism, it is
of interest to determine whether zebrafish MAO is functionally
more similar to human MAO A or to MAO B. Finally, the develop-
ment of a robust expression system facilitates the use of mutagene-
sis to probe the structural and functional properties of MAO A and
MAO B.
An initial report of human MAO A and MAO B heterologous
expression [4] in Saccharomyces cerevisiae showed the expressed
enzymes to be mitochondrial localized and to exhibit activities
consistent with their known properties. Unfortunately, the levels
of enzyme expression did not permit purification of large quantities
of enzymes suitable for physical and biochemical investigation.
Weyler [5] published a S. cerevisiae expression system that did
provide high levels of human MAO A. Unfortunately, for unknown
reasons, this expression system does not result in large quantities of
human MAO B when attempted in our laboratory. Human MAO B
(as well as human MAO A) was shown to be successfully expressed
in reasonable levels in the insect cell system [6] and is the source of
commercial (Sigma-Aldrich) membrane preparations of these
enzymes.
Given the current information on the heterologous expression
of human MAO A and MAO B, we initiated an investigation of
Pichia pastoris (Fig. 1) as a possible expression system for these
membrane-bound enzymes. This system exhibits a number of
favorable characteristics leading to success in producing high levels
of MAO. Pichia is grown on glycerol with an aerobic fermentation
and high mitochondrial production. The foreign gene is
incorporated into the genome of the organism providing a stable
expression system. Gene expression is under a methanol-inducible
promoter; thus, the growth and expression require only glycerol,
salts, and methanol which are inexpensive components of a growth
media. Pichia can be grown to high cell densities so large quantities
of cell mass can be grown in relatively small volumes of media
(2–5 L) when fermentation is carried out under pH control and
high levels of aeration. As described in this article, human MAO A
and MAO B [7, 8] (see Note 1), rat MAO A [9] and MAO B [10],
 Recombinant MAO Expression and Purification 13

Fig. 1 Pichia pastoris colonies on an MD plate

and zebrafish MAO [11] can be expressed and purified in 100 mg

quantities. The enzyme preparations are all expressed in their
mitochondrial-bound forms, contain covalent FAD cofactors, and
are fully active in their respective purified forms. Purification pro-
cedures for all of the expressed enzymes described here are identical
with regard to membrane preparations and enzyme solubilization.
Differences in column purifications of the enzyme detergent
extracts have been identified and are described here. It should be
noted that no affinity tags to assist in purifications are used in our
procedures and most of the work in our laboratory was performed
on untagged MAOs. In the case of human MAO A, a high-
resolution crystal structure was published by another group which
was based on recombinant human MAO A expressed in Saccharo-
myces cerevisiae as an N-terminal His6-tag protein [12]. More
recently, Binda and collaborators (including the author of this
chapter) carried out a biochemical characterization of human
MAO A mutants expressed in Pichia pastoris as an N-terminal
His6-tag protein [13]. The reader can refer to the methods of
these articles for the affinity chromatography purification proce-
dures of His-tagged forms of MAO A.
14 Dale E. Edmondson

2 Materials

2.1 Plasmid Source All plasmids that we have constructed for expression of MAO in
Pichia pastoris have been deposited in the NIGMS-sponsored Pro-
tein Structure Initiative (PSI) Repository in Tempe, Arizona
(http://psimr.asu.edu). Plasmids can be acquired from them for a
nominal fee. To assist the reader in the acquisition process, the
identification number for each of the pPIC3.5 K Pichia expression
vectors containing the respective MAO genes is as follows:
Human MAO A: Hs CD00429730.
Human MAO B: Hs CD00429731.
Rat MAO A: Rn CD00423479.
Rat MAO B: Rn CD00423480.
Zebrafish MAO DR CD00443481.
The sequence for the MAO gene in each clone has been verified
both in our laboratory and by the PSI. Each vector has been
successfully used for the preparation of expression strains of Pichia
pastoris KM71 (commercially available from Invitrogen). Con-
structs of expression systems can be prepared using either sphero-
plasts or the electroporation procedures. In our hands, the
spheroplast method results in higher expression levels. It should
be noted that the use of the spheroplast method requires extreme
attention to sterile conditions to avoid contamination. Methodol-
ogy for either method of construct preparation is given in the
Invitrogen manual [14] and followed in our laboratory.

2.2 Media and The reader can refer to the Invitrogen manuals for protein expres-
Reagents for Pichia sion in Pichia pastoris that are freely available online. Instruments
pastoris Cell Cultures required to grow Pichia pastoris are glass flasks (1–5 L) and
temperature-controlled aerated and shaking incubator. For large-
scale cultures, a New Brunswick BioFlo 3000 fermenter is advis-
able. For fermentation, autoclavable feeding tubes must be used
(e.g., Nalgene cat # 8000-9020) and it is advisable to have an
industrial oxygen tank to supply air with oxygen. A solution of
50% NH4OH (prepared with distilled water) is generally used to
control pH and 100% methanol is necessary to induce MAO
expression.

2.3 Buffers and 1. Cell breaking buffer: 50 mM sodium phosphate at pH 7.5, 5%

Reagents for glycerol.
Purification of 2. TEA buffer: 0.1 M triethanolamine/HCl, pH 7.2.
Recombinant MAOs
3. Detergent extraction buffer: 10 mM potassium phosphate,
pH 7.2.
4. Buffer MAOA/glyc-A: 10 mM potassium phosphate, pH 7.2,
20% glycerol (for human/rat MAO A and zebrafish MAO).
 Recombinant MAO Expression and Purification 15

5. Buffer MAOA/glyc-B: 250 mM potassium phosphate, pH 7.2,

20% glycerol (for human/rat MAO A and zebrafish MAO).
6. Buffer hMAOB: 25 mM potassium phosphate, pH 7.2 (for
human MAO B).
7. Buffer hMAOB/glyc-A: 25 mM potassium phosphate, pH 7.2,
20% glycerol (for human MAO B).
8. Buffer hMAOB/glyc-B: 250 mM potassium phosphate,
pH 7.2, 20% glycerol (for human MAO B).
9. Buffer rMAO B: 25 mM potassium phosphate, pH 7.6 (for rat
MAO B).
10. Buffer rMAOB/glyc-A: 25 mM potassium phosphate, pH 7.6,
20% glycerol (for rat MAO B).
11. Buffer rMAOB/glyc-B: 250 mM potassium phosphate,
pH 7.6, 20% glycerol (for rat MAO B).
12. Storage buffer: 50 mM potassium phosphate buffer, pH 7.2
(human MAO B) or 7.6 (rat MAO B), 20% (v/v) glycerol,
0.8% (w/v) β-octyl-glucopyranoside.
13. β-octyl-glucopyranoside powder.
14. Triton X-100.
15. Calcium chloride solution: 1 M CaCl2.
16. Biuret reagent to measure protein concentration.
17. Phospholipase A2 and phospholipase C.
18. Ammonia solution: 2 M NH4OH.
19. Dithiothreitol (DTT) powder.
20. Ethylenediaminetetraacetic acid (EDTA) powder.
21. Phenylmethylsulfonyl fluoride (PMSF) powder.
22. Benzylamine (MAO B) and kynuramine (MAO A and zebrafish
MAO) powders.
23. d-Amphetamine powder.

2.4 Instruments and 1. UV/Vis spectrophotometer (to determine protein concentra-

Tools tion by biuret assay and to detect protein during purification).
2. FPLC system (although chromatographic runs can be also
carried out by gravity).
3. Vortex mixer.
4. Silica/zirconia beads 0.5 mm (Biospec BeadBeater, Bartles-
ville, OK, USA).
5. Stainless steel BeadBeater (Biospec BeadBeater, Bartlesville,
OK, USA).
6. Miracloth (Calbiochem).
7. DEAE-Sepharose column (25
600 mm) (for MAO A and
zebrafish MAO).
16 Dale E. Edmondson

8. Ceramic hydroxyapatite column (for zebrafish MAO).

9. Bio-Rad Macro-Prep® High-Q anion-exchange column (for
MAO B).
10. Amicon Ultra 30 K centrifugal filter (Millipore).
11. Handheld homogenizer or motor-driven glass/Teflon homog-
enizer (see Fig. 1.1 in Chap. 1).
12. High-speed centrifuge (up to 150,000 g).

3 Methods

3.1 Construct Selection of expressing cells is performed by an initial screening for

Preparation the His+ phenotype as described in the Invitrogen manual. Further
selection of high-level expression strains indicating multiple gene
insertions is performed using the resistance to the antibiotic G-418
in concentration ranges of 0.25 mg/mL to 1.25 mg/mL:
1. Select G-418-resistant colonies that can be detected after
4 days at 30 C. Several G-418-resistant colonies are selected
and grown in shake flasks (see Subheading 3.2).
2. Cells from the shake flask cultures are collected and broken
with glass beads by vortex in a test tube. Following low-speed
centrifugation to remove glass beads, unbroken cells, and large
cell debris, the supernatants are assayed spectrophotometrically
for MAO activity using the kynuramine oxidase assay [15] for
MAO A or zebrafish MAO or benzylamine oxidation for MAO
B [16] (see Note 2). Cultures exhibiting the highest level of
specific activity are used for preparation of stock cultures (see
Invitrogen manual) and stored at 80 C in 20% (v/v) glycerol.

3.2 Small-Scale The reader can refer to the Invitrogen manuals (freely available
Growth of Pichia online) for small cell cultures of Pichia pastoris that are made in
pastoris Cultures flasks.

3.3 Large-Scale Optimal growth and high-level expression of MAOs in P. pastoris is

Growth of P. pastoris- performed using a fermenter equipped with pH control, O2 moni-
Expressing Strains toring, and the ability to sample the fermentation media (see Note
3). Our laboratory has routinely used the New Brunswick BioFlo
3000 fermenter equipped with 2-L or 6-L reactor vessels. It is
important to underline that the culture volume increases signifi-
cantly with respect to the initial volume; thus, it is advisable to start
with about 60–70% of the vessel capacity. All parameters and
feeding flow rates indicated below are referred to liters (L) of the
initial volume. The Invitrogen website also includes manuals for
Pichia fermentation. Herewith, the protocol adapted for MAO
expression is described.
 Recombinant MAO Expression and Purification 17

1. One week earlier, streak a MAO glycerol stock on a MD plate

(Invitrogen manual) and incubate at 30  C for 2–3 days
(Fig. 1). Autoclave 1 L of distilled water distributed in bottles
(250 mL each), 1 L of 50% glycerol, 300 mL MGY medium
(Invitrogen manual), three fermenter feeding tubes. Prepare
100 mL PTM1 trace salts (Invitrogen manual).
2. Autoclave fermenter with the basal salt medium (Invitrogen
manual) corresponding to the culture initial volume. Supple-
ment the sterilized medium with 12 mL/L of trace salt solu-
tion (Invitrogen manual) immediately before inoculation with
5–10% of the initial fermentation volume (e.g., ~300–400 mL
for 6 L medium) of a Pichia culture grown in MGY medium
(OD600 ¼ 2–6; it may be useful to pre-inoculate a colony into
20–30 mL of MGY, leave it growing overnight, and use this
small culture to make the second pre-inoculum of 300–400 mL
mentioned above). Leave cells growing at 30  C for about
20–24 h. Dissolved oxygen concentration in the growth
media is maintained at 30–100% of air saturation either by
adjusting the agitation rate or by supplementation with an
air-oxygen mixture through an automated regulator.
3. Start glycerol feeding when the wet cell weight reaches 100 g/
L: a 50% (v/v) glycerol water solution (supplemented with
12 mL/L trace salt) is fed to the media at an initial flow rate
of 18 mL/L/h. Glycerol feeding is stopped when the cell
density in the culture doubles (wet cell weight ca. 200 g/L).
4. Start induction of MAO expression by 100% methanol (sup-
plemented with 12 mL/L trace salt solution) feeding. The flow
rate of the methanol feeding is adjusted slowly to a final flow
rate of 3 mL/L/h. At this point, aliquots of 1 mL of cell
culture were taken out every 12 h and assayed for MAO expres-
sion levels. A maximum expression level is achieved after
ca. 80–96 h of methanol induction (zebrafish MAO is an
exception where maximal expression occurs after 24 h) (see
Note 4).
5. The cells are harvested by centrifugation (20 min) at 1500
g
and washed once with the cell breaking buffer with 1 mM
PMSF and 1 mM EDTA added upon usage. The washed cell
pellets are combined into aliquots used for subsequent protein
purification, frozen in liquid nitrogen, and stored at 80  C.

3.4 Preparation of The following protocol can be used for either recombinant MAO A
Membrane Particles or MAO B. After each centrifugation, the pellet is better resus-
Containing MAO pended by using a handheld or motor-driven glass/Teflon homog-
Activity (See Note 5) enizer (see Fig. 1.1 of Chap. 1).
1. The frozen cell paste from 1 L of fermentation is thawed and
resuspended in ~400 mL cell breaking buffer containing 1 mM
18 Dale E. Edmondson

PMSF, 1 mM EDTA, and 30 μM DTT added upon usage, at

4 C for 4 h (to speed up the procedure, the cell paste can also
be thawed overnight on ice).
2. After the pellet is mostly thawed, fill small stainless steel Bead-
Beater canisters approximately half full with 0.5-mm silica/
zirconia beads. Add the cell suspension. Break (in 4  C cold
room) with six cycles of 2-min beating and 8-min cooling in an
ice/salt bath. Carefully pour the broken cell/glass bead sus-
pension into a separate container. Use a timer to maintain the
schedule for each cycle (see Note 6).
3. After breaking, the glass beads are separated from the cell
homogenate by filtration through a layer of Miracloth (Calbio-
chem). The separated glass beads are then washed with a small
volume of the cell breakage buffer and can be reused with a new
volume of cell suspension (at the end beads can be washed with
water/soap, rinsed, and dried; they can be used for a number of
preps but it is advisable to get new ones when cell disruption
efficiency decreased).
4. Unbroken cells and large cell debris are removed from the
lysate by centrifugation at 1500
g for 20 min at 4  C.
Remove the supernatant, measure the volume, and freeze at
least two 1-mL aliquots for subsequent activity and protein
analysis.
5. The membrane fraction is separated from the soluble compo-
nents of the cell lysate by centrifugation at 100,000
g for
30 min at 4  C. The supernatant is discarded and the pellet is
resuspended to the desired protein concentration in 0.1 M
TEA (triethylamine hydrochloride) buffer at pH 7.2. Measure
the volume and activity of the resuspended high-speed mem-
brane particles. Usually at least 60% activity can be recovered
for MAO A and at least 75% activity can be recovered for MAO
B. The particles are ready to use in subsequent steps or they can
be stored in 20% glycerol at 80 C for several months.

3.5 Extraction and The following protocol can be used for either recombinant MAO A
Solubilization of MAO or MAO B. After each centrifugation, the pellet is better resus-
from Membrane pended by using a handheld or motor-driven glass/Teflon homog-
Particles enizer (see Fig. 1.1 of Chap. 1).
1. The membrane particles containing MAO activity are sus-
pended to a protein concentration of 25 mg/mL in TEA
buffer. The suspension is then digested by the addition of
1 mg of phospholipase C and 6700 units of phospholipase A2
per 500 mg of protein in the presence of 25 mM CaCl2. The
digestion reaction is stirred at room temperature in the dark for
1 h with the pH maintained at 7.0–7.2 by the addition of
ammonia solution.
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things the first is more important, in others the second. For example,
under normal conditions the height of a man or woman is determined
almost altogether by inheritance, for no one by taking thought can
add a cubit to his stature. Intelligence, likewise, is to a large degree
an inherited quality. But morals, manners, education, and personal
habits are determined much more largely by environment than by
inheritance.
The respective influences of inheritance and Which is the more
environment cannot, however, be in all cases potent influence?
clearly separated. Both often work to produce the same result, as
when a person who inherits a strong body and a sound mind is
fortunate enough to be placed in an environment where both body
and mind are developed by out-of-door life and a good education.
Sometimes they work in opposite directions, as when a child starts
life with a strong physique and good natural intelligence, but grows
up in a crowded tenement amid sordid conditions which weaken the
one and fail to afford scope for the other. We cannot say, therefore,
that one factor is always stronger or weaker than the other. The
social progress of the race is promoted by improving both influences.
Environment especially can be improved by human effort. Man’s
control of his inheritance is not nearly so complete, but everything
that conduces to the betterment of health or education and promotes
a higher morality is a step towards improving its influence.
Physical and Social Inheritance.—The The two forms of
influence of inheritance is exerted from two inheritance.
quarters which may be distinguished by calling them physical and
social. By the former we mean the influence (a) Physical.
exerted upon human beings by the bodily and
mental traits which are handed down to them by their own parents.
Not all the characteristics of parents are transmitted to their children
but mainly those which the parents themselves have inherited. Traits
or qualities which have been acquired by the parents during their
own lives do not ordinarily descend to their children.[5] Parents who
are born feeble-minded will in all probability have feeble-minded
children; but parents who acquire through education a high degree of
learning and culture cannot transmit any of this to their children by
inheritance. There is no royal road to learning. Some of us are born
with better or worse possibilities than others, but we are all born
illiterate.
The other form of inherited influence is called (b) Social.
our social heritage because it represents the
whole accumulation of knowledge, habits, and expedients which
have come down to us by the social process of teaching and
learning. Each generation of mankind is enormously dependent upon
its social inheritance; without it everything that we now call
civilization would collapse in a very short time. Each generation
takes over all the knowledge possessed by the one which went
before; each generation adds something to this stock of knowledge,
habits, and expedients for the benefit of the generation which comes
after it. Each generation, if it is to live happily, must adapt this social
inheritance to its own particular needs.
Physical and Social Environment.—The The two kinds of
other great influence is that of environment. By environment:
physical environment we mean the conditions of nature and society
in which man lives, moves, and has his being. Physical environment
includes the geographic, climatic, and other natural conditions which
surround the people. These conditions have an (a) Physical.
important influence upon the trend of human
development and they are not, for the most part, under man’s
control. Man must adapt himself and his ways of life to them. In cold
climates he must wear warm clothing, provide artificial heat in
houses, and consume warmth-giving food. Groups of men must
everywhere mould their occupations to the character of the soil, the
natural resources, and the other conditions of the physical
environment in which they live. It is because of differences in
physical environment that the Southern states developed cotton-
culture on a large scale and employed slave labor, while the
Northern states gave their attention to farming and industry with free
labor.
Physical environment, moreover, determines in some measure the
relations of the various races with their neighbors. Men will be
influenced by neighboring groups of men in so far as physical
features make intercourse easy or difficult. A race of men who live
on a distant island, or in any other shut-off region, will not be so
easily influenced by neighboring races as if they dwelt in the midst of
a fertile plain. To some extent, as has been said, man is able to
overcome the difficulties which physical environment sets in the face
of progress. If there is inadequate rainfall, he may devise a system of
irrigation and carry on certain forms of agriculture as successfully as
though rainfall were abundant. By means of railroads, steamships,
and electric or radio communication he can be in constant contact
with other men who are separated from him by physical obstructions.
But however much the conditions of nature may be controlled, they
still exert a great influence upon human progress.
The social environment is quite a different (b) Social.
thing. By it we mean the conditions altogether
apart from geographic or natural features, which influence the daily
life of mankind. We include within social environment such things as
family life, the schools, the churches, the organization and methods
of industry, the form of government—everything that society
develops in the way of institutions. Many of these, as has been
pointed out, are natural growths, but the mind of man has also had a
large part in shaping their course.
Most of the things we do, whether as a body How customs and
of people or as individuals, are merely the result laws create a social
of custom or general habit. Why do men have environment.
their hair cut short while women let their hair grow long? Why do
people wear black when they are in mourning? In some countries
they wear white. The answer is merely that every nation, through
long-continued habit, develops its own ways of doing things and
keeps on doing things in that way regardless of any present reason.
Orientals, when they eat their meals, squat on the floor; Europeans
and Americans seat themselves at the table. Aryans shake hands
when they meet; the Esquimos hold their hands high above their
heads as a token of greeting. The gentleman of today, when he
greets a lady on the street, raises his hat. This is not a particularly
graceful custom, nor is it in rainy weather an altogether hygienic one;
but it has been in vogue among the people of western Europe for
many centuries. It goes back to the days of chivalry when the
armored knight raised his visor to show his countenance and
disclose his identity.
 Primitive races are governed largely by customs, and not until a
race has shown itself amenable to the influence of custom is it
prepared to be governed by laws. Laws differ from customs in that
they have a definite sanction, in other words are enforced by some
official authority. The institutions and practices which make up the
social environment may be the outcome of long-standing custom,
like the system of trial by jury, for example; or they may be brought
into existence by law, as, for instance, the admission of women to
suffrage or the establishment of national prohibition. The avowed
purpose of all human institutions is to promote the greatest good of
the greatest number, in other words to provide the best social
environment.
Some Important Social Forces.—The basis of custom is habit.
Customs, in other words, are habits which extend to the whole
community and receive its approval. We do not Two important
always realize how great a part habit plays in social forces: habit
our daily lives.[6] Without it the day’s work could and imitation.
not be done. By habit we walk, eat, dress ourselves, and perform
many other common acts. Just think how long it would take a novice
to put the various parts of a watch together; but the watchmaker,
being habituated to the task, can do it in an hour. The foundation of
habit is imitation. One man does a thing successfully; others follow
his lead; a habit develops and a general practice or custom may be
the ultimate outcome. The influence of custom is usually
conservative, for when a custom is once firmly established it does
not easily give way. Take the custom of smoking tobacco, for
example. Europeans found it in vogue among the Indians when they
first came to America; they adopted it and have kept it up for more
than four hundred years. Sometimes, however, the habit or custom is
only of short duration, in which case we commonly call it a fashion.
Fashions come and go. A century ago men used snuff and women
powdered their hair; but these things have wholly passed out of
fashion today.
The Course of Social Progress.—Having considered the various
social factors and forces (development, inheritance, environment,
custom, and so on) we are now in a position to ask and to answer
the following question: In accordance with what principle has human
society developed?
There was a time when even educated people imagined that such
organizations as the state were planned in advance, that individuals
merely came together in prehistoric days and agreed after calm
deliberation to establish a civil government. That, of course, was an
absurd idea. Today we realize that one step in social organization led
gradually to another, that institutions were not created but evolved,
that various social factors and forces exerted an influence upon their
development, and that the strongest institutions survived while the
weaker disappeared.
In the course of human history associations of Institutions that
every type have come into existence; many still have succumbed in
continue to flourish while others merely abode the struggle for
their little hour and went their way. The existence.
organizations which we have today, including the family, the school,
the church, the community, the state, are among those that have
survived. Those that succumbed during the long journey down the
ages would make a formidable list. Who ever hears nowadays of the
totem-kin, the clan, or the gens? Where do we now find tribunes,
praetors, augurs, and triumvirates? Absolute monarchy, as a form of
government, once held sway over most of the world. But democratic
government entered into competition with it, and as there was not
room enough for both, one crowded the other off the stage. The
great mediaeval institution of feudalism dominated the rural life of
Europe for more than five hundred years, but the last relics of feudal
tenure have practically all been swept away. The trade guilds of the
olden days, the orders of nobility, the crowns and coronets, the
soothsayers and the alchemists—all of them have disappeared or
are rapidly disappearing. The beaches of history are strewn with the
wrecks of social and political institutions. Some others, like the
hereditary peerages of a few European countries, are barely able to
keep afloat. The institutions which survive and flourish are the ones
that have been found best fitted to survive.
The Fundamental Social Group.—In human Importance of the
society the foundation-group is the family. family.
Human beings are social by nature; the motive which draws people
together is so universal that we call it a natural instinct. Individuals
do not live in isolation. Nobody leads the hermit type of life if he can
avoid it. Robinson Crusoe was not on his little island because he
wanted to be there. Even among the least civilized races of men,
among savage tribes, there is a grouping of men, women, and
children on the basis of blood relationship. The family, as a unit, is
older than either the state or the community. It is the foundation upon
which other groups and organizations have been built, hence it is
rightly called the “social microcosm” or basis of society.
The primary function of the family is to keep The function of the
the human race in existence. Its first duty is the family.
rearing of children so that a new generation may take the place of
the old. Other duties that belong to the family may be handed over to
the school (the duty of secular education) or to the church (the duty
of religious instruction); but the primary function of the family, that of
perpetuating the race, is one which cannot be transferred.
The whole stream of human life flows through the family
organization. The same virtues which make for harmony in the
household,—obedience, co-operation, loyalty, and service,—are the
ones which mark good citizenship; therefore the home is the primary
school of all the civic virtues. For this reason the collapse of the
home and of home life would be nothing short of a human
catastrophe.
 THE FAMILY. By Charles Sprague Pearce
From a Copley Print, copyright by Curtis & Cameron, Boston.
Reproduced by permission.

THE FAMILY

By Charles Sprague Pearce

From the mural painting in the entrance pavilion of
the North Hall, Library of Congress.
This is the ancient family. The father has just
returned from hunting. The mother holds up the baby
to welcome him and his little daughter throws her arms
about him. On the benches of stone at either side of
the group sit the grandmother and grandfather, the
latter with the air of a patriarch. By his side lies a scroll.
Three generations are placed together in the idyllic
environment of which the poets have so often written.

Other Social Groups.—Out of the family The clan.

grew the clan, or group of families united by ties
of kinships. In early days the clan was a wandering group, like the
gipsy bands of today; but ultimately each clan settled upon the land
and became a community. In these communities men became
trained in manual labor; they developed customs and the rudiments
of a village government.
Then came the next stage—several village communities joined
together for defence against their mutual enemies. A loose
confederation at first, this group of communities in time became a
state, usually with a chieftain or a monarch at its head. In other
cases a single village community grew in size to such an extent that
it became a city state, like Athens or Sparta. The most notable
example of this development is afforded by ancient Rome, where a
small community grew into a World Empire. The family, the clan, the
community, the confederation of communities, the state, and,
ultimately, the nation—that is the general course of political
evolution, although this line of development was not in all cases
exactly followed.
But the evolution was not confined to political institutions alone.
Social and economic groups and institutions developed also. A
variety of needs called forth one institution after another, the church,
the school, the club, the industrial organization. Each has its own
function in organized society.
The Rôle of Government.—The dominating Government is the
factor in the social and economic life of today, guiding hand.
however, is the political community (nation, state, and municipality),
acting through the agency which we call government. Government is
the great co-ordinating factor. Day by day we are looking more and
more to government for leadership, for regulation, and for
supervision in all our greater social and economic activities. In the
field of social and economic effort, all roads lead through
government—it is the clearing house of our greater problems.
Whether the problem be one of banking, commerce, poor-relief,
labor, or defence we must reckon with the hand of government as
one of the strongest among constructive and harmonizing factors.
Government is the focal point in all civic relations. It provides a
thread which winds its way along every main line of civic activity.
That has been particularly noticeable in European countries; but it is
now true of America as well. The greatest of our socializing agencies
is government.
Individual Liberty and Social Control.—In America’s emphasis
the United States strong emphasis has always on individualism.
been laid upon individual freedom, and rightly so, because the
encouragement of individual initiative is essential to progress in a
new country. The exploitation of vast natural resources required that
men should be given encouragement to pioneer, and should not be
held down by too much governmental interference. “That
government is best which interferes the least” was the common
notion. Stress was laid upon the prosperity of the individual rather
than upon the welfare of the whole people. This doctrine of extreme
individualism undoubtedly served a useful purpose in the days when
the country’s biggest problem was to increase production and gain
for itself a place among the strong nations of the world.
To a considerable extent this emphasis upon The influence of the
individualism was due to the influence of the frontier.
frontier. From the first settlement of the country down to about 1880
the American people were engaged in the task of marching steadily
westward, conquering the wilderness as they went. This mastering of
a great domain demanded qualities of enterprise, initiative, and
individual courage. It developed men’s confidence in their own power
and made them reliant upon their own efforts. In old countries the
natural tendency is for the individual to look to the public authorities
for leadership, guidance, and supervision; on the great American
frontier the pioneers had to hew their own way. They preceded the
state and the community. This emphasis upon individual initiative
remained as the frontier rolled west and profoundly affected the
whole social temper of the country.
In time a reaction came. Social control The nature and
developed. The more thickly populated a country scope of social
becomes, the more complicated do the relations control.
of individuals grow, and the greater is the need for general restraint.
Social control, however, is not merely negative in its purpose. Its
object is not simply to restrain individuals in their freedom of action
but to encourage them in the thing which the general welfare
demands. The government is the chief agency through which social
control is exercised, but it is by no means the only one. Religious,
fraternal, professional, and benevolent organizations do a good deal
in the same general direction. Their function is to promote collective
interests as distinguished from the interests of individuals; they
protect the collective interests against the avarice or selfishness of
individuals. The government exercises social control by means of
laws and administrative orders; other organizations exercise it by
their own rules or by customs which the members obey.
There is always a danger, of course, that The limits of social
social control may proceed too far. It is not the control.
object of government and of social organizations to run all men in the
same mould, making them mere automatons without individuality or
initiative. Government should aim to give sufficient scope for every
individual to use his abilities in the best possible way. Control over
the acts and discretion of individuals is justified only where such
control promotes, in the long run, the well-being of the greatest
number of individuals. The state is not an end in itself. Society is not
an end in itself. The individual is the end. Society and the state are
merely means to the promotion of the general welfare and the
welfare of the individual. Their activities in the way of exercising
social control should go no further than this.
General References
C. H. Cooley, Social Organization, pp. 3-22;
H. G. Wells, Outline of History, Vol. I, pp. 3-103;
C. A. Ellwood, Sociology and Modern Social Problems, pp. 7-59;
F. S. Chapin, Social Evolution, pp. 3-101;
Vernon Kellogg, Darwinism Today, pp. 10-57; 129-157;
J. A. Thomson, Darwinism and Human Life, pp. 181-237;
D. S. Jordan and V. L. Kellogg, Evolution and Animal Life, pp. 1-56;
T. N. Carver, Sociology and Social Progress, pp. 174-270;
E. A. Ross, Social Control, pp. 1-105.
Group Problems
1. How far should society control the conduct of individuals? Why social
control is exercised. The extent of social control in older countries,—Great Britain,
France, and Germany. Its growth in America. Causes of this growth. The point at
which it ceases to be justified. Illustrations. Effects of too much emphasis on
individualism. Effects of too much social restraint. Relation of social control to
socialism. References: E. A. Ross, Social Control, pp. 49-76; H. G. Wells, New
Worlds for Old, pp. 1-55; T. N. Carver, Sociology and Social Progress, pp. 788-
808; Ibid, Principles of National Economy, pp. 740-749.
2. What is progress? References: F. S. Marvin, Progress and History, pp. 8-
10; John Dewey “Progress” in International Journal of Ethics, xxvi, 312-318
(1916); James Bryce, Essays and Addresses in War Time, pp. 84-102 (War and
Progress); George Nasmyth, Social Progress and the Darwinian Theory.
Short Studies
1. The past in the present. H. R. Burch and S. H. Patterson, American
Social Problems, pp. 33-43.
2. The beginnings of civilization. H. G. Wells, Outline of History, Vol. I, pp.
183-208.
3. Earlier forms of the family. C. A. Ellwood, Sociology and Modern Social
Problems, pp. 108-130.
4. The development of the tribe into the community. J. Q. Dealey, The State
and Government, pp. 24-45.
5. The American family as an economic unit. Mary K. Simkhovitch, The City
Worker’s World, pp. 1-21.
6. The relation of leisure to family life. Florence Kelley, Some Ethical
Gains through Legislation, pp. 105-125.
7. The influence of environment. F. S. Chapin, Social Evolution, pp. 121-170.
8. Habit. William James, Psychology, I, pp. 104-127.
9. The influence of frontier conditions upon the development of American
society. F. J. Turner, The Frontier in American History, pp. 1-38.
10. Individualism. Herbert Spencer, Social Statics, pp. 100-136; C. W.
Eliot, The Conflict between Individualism and Collectivism in a Democracy, pp. 1-
42.
Questions
1. How would you define “society”? Is it an organism? What resemblances to an
organism does it bear?
2. Explain the following terms: instinct; impulse; natural selection; social
inheritance; social environment; habit; custom; fashion; mob mind; institution;
social control. Give examples of each.
3. How has physical environment affected the ideas of the American people in
relation to (a) national defence; (b) form of government; (c) social control? How
has the physical environment of England affected the ideas of the English people
on the same matters?
4. What racial characteristics do you find most strongly marked in the (a) Scotch;
(b) Irish; (c) Scandinavians; (d) Italians; (e) Jews; (f) Japanese?
5. Name any institutions, other than those given in the text, which have served
mankind for a time and been discarded.
 6. Can you think of any customs which are universal throughout the world? Any
which prevail in the United States but not elsewhere? Any which prevail in some
parts of the United States but not in others?
7. Do Americans in general pay too much deference to custom? Is there any
ground for the European idea that there is “too much uniformity” in American life?
8. Are crowds likely to be more conservative or more radical than the individuals
who compose them? Give your reasons. What is the difference between a mob, a
crowd, a meeting, and a deliberative assembly?
9. The family, as an organization, differs not only in size but in nature, from all
other social organizations such as the community, the state and the nation. Show
how this is.
10. In what ways would society suffer if the family as a social unit were broken
down?
11. To what extent has society the right to regulate, as a measure of self-
protection, the institution of marriage?
12. Why have we laid emphasis upon individualism in this country? Explain why
this stress is being steadily diminished.
13. Should social control be exercised over (a) the methods of agriculture; (b)
the marketing of timber; (c) the production and sale of tobacco; (d) the rates
charged by electric lighting companies; (e) the price of bread at retail bakeries; (f)
the hours of labor for men; (g) the hours of labor for women; (h) the kind of
pictures shown in theaters; (i) the diet of the people; (j) the hours at which young
people may be on the streets after dark; (k) the religious beliefs of the people? Tell
why or why not in each case. Is a greater degree of social control justified over
certain classes of the population than over others? Is it justified at certain times
and not at others?
14. If society exercises too little control over the individual, what evils result? If it
exercises too much control, what are the consequences (a) upon the individual; (b)
upon society itself?
Topics for Debate
1. Physical environment has had a more important influence than racial
characteristics in determining the establishment and maintenance of democratic
institutions in the United States.
2. Thomas Jefferson was right when he said “That government is best which
governs least”.
3. Society is under obligation to ensure every industrious man a decent living.
 CHAPTER II
THE PEOPLE, RACES, AND RACIAL PROBLEMS
OF
THE UNITED STATES

The purpose of this chapter is to explain how the population of the United
States has grown, how it is distributed, the varied races of which it is
composed, and the racial problems which immigration has created.

We, the People of the United States.—The How the national

national constitution begins with the words: “We, population has
the People of the United States”. But who are grown.
the people? No one cares to carry a lot of figures in his head and
most people think that statistics are uninteresting; but there are
some figures which everyone ought to know and, moreover, there
are some statistics which are far from being dry or tedious. The
figures relating to the population of the United States, its growth and
distribution, are interesting because they portray something that the
world has never seen before and probably will never see again,—a
country doubling its population four times in just about a single
century. That is what the United States did in the hundred years from
1790 to 1890. In 1790 the entire population was less than four
millions. (There are more people in the single city of New York today,
and almost as many in Chicago.) The population had doubled in
1813, and had doubled again in 1838, when it passed sixteen
million. In 1864, only twenty-four years later, it had doubled once
more (32,000,000). It passed the sixty-four million mark in 1891, and
in all probability will double for the fifth time about the year 1940. In
1920, when the last national census was taken the population had
passed 105,000,000.
Now if you will take the data at the foot of the Will this increase
page and work the calculation to the year 2000 continue? If so, with
(allowing for the gradual slackening of the what results?
increase) what will the population of the United States be then?[7] It
will be around three hundred millions, which is more than the white
population of the entire world today. Even so the country would not
be nearly so thickly populated as some European countries now are.
Today there are in the United States about thirty-five persons to the
square mile. But there are 700 to the square mile in England and
658 per square mile in Belgium, which are two of the most thickly-
populated of all countries at the present time.[8] Three hundred
million people in the United States, if the figure should reach that
total at the end of the twentieth century, would be less than one
hundred per square mile. There would still be six acres of ground for
every man, woman and child in the country. Whether there would be
a sufficient food supply to support so large a population is another
question and one which is not so easily answered.

THE CENTER OF POPULATION

The way in which the center of population has been
steadily moving westward is indicated by this map. It
will be noted that in its journey to the west it has held
closely to the 39th parallel of latitude. The median
point, on the other hand, has remained practically fixed
during the last thirty years.
The location of the median point is determined as
follows: Take the parallel of latitude which divides the
country in such manner that half the population is north
of that parallel and half is south of it; similarly, take the
meridian of longitude which divides the country in such
manner that half the population is east of it and half is
west of it; the intersection of this parallel and this
meridian is called the median point.
 CENTRE OF POPULATION
AT EACH CENSUS: 1790 TO 1920

MEDIAN POINT
1880 TO 1920

☆ CENTER OF POPULATION Δ MEDIAN POINT

How the National Population is Distributed. Some areas are

—The people of the United States are very more densely
unevenly distributed over the face of the country. populated than
others.
Great areas, particularly in the West, have only a
few persons to the square mile, while the crowded sections of the
largest cities have many hundreds to the acre. Even the rural areas
of some states are thickly settled while in others the people are few
and far between. Rhode Island is nearly as densely populated as
Belgium, having about 566 people to the square mile while many
states of the Western mountain region have only seven or eight
inhabitants per square mile of territory. The reasons for this uneven
distribution are chiefly geographical. The states which have grown
most rapidly in population are not necessarily the oldest states but
the ones which have the greatest natural advantages in the way of
fertile soil, or mineral resources, or harbors and waterways, or
favorable climate. People make their homes in those regions where
they can best make a living.
Favorable climatic conditions exert a strong Climate affects the
influence in attracting population. It is an density of
interesting fact, often noted by students of population.
history, that whereas civilization developed earliest in the tropical
and semi-tropical zones it has everywhere made its greatest
advance in the regions of moderate temperatures and rainfall. Taking
the broad strip of country which lies between the thirty-seventh and
the forty-fifth parallels of latitude, it will be found that nearly four-fifths
of the entire population is concentrated within this mid-latitude area
of the United States. And this is the area which, in all probability, will
continue to be the most thickly settled.
The center of population in 1920 was at The center of
Whitehall, Owen County, Indiana, a little town of population in 1920.
forty-three inhabitants which burst into prominence overnight when
the census bureau announced it as the pivot of the nation. By the
center of population is meant the point which the greatest number of
people could reach with the least amount of travel. This point, at
each successive census, has been steadily moving westward
following very closely the thirty-ninth parallel of latitude. In 1790 it
was at Baltimore, in 1840 in West Virginia, in 1880 in Ohio, and since
1890 it has been moving westward across Indiana.[9] The center of
population is a long way from the center of area, the latter being in
Northern Kansas not far from the Nebraska border.
The Drift of Population to the Cities.—The distribution of the
people has also been influenced by the growth of large cities. In
1790 there were only five communities with populations exceeding
8000, namely New York, Philadelphia, Boston, Baltimore, and
Charleston. Taken altogether they had only 130,000 inhabitants, that
is to say only one person in every thirty lived in these towns. But so
rapidly did the various towns and cities spring up all over the country
that by the time of the Civil War there were nearly 150 with
populations above 8000, and today there are nearly a thousand. Not
a single American city had 70,000 population in 1790; today there
are more than a hundred such cities.
This remarkable drift of population into the The chief causes of
towns and cities, which began early in the city growth.
nineteenth century and has continued ever since, shows no signs of
slackening. It is due to many causes, including the great demand for
industrial labor in the cities, the attractiveness of city life to the young
men and women of the country districts (see p. 351), and the
tendency of the immigrants to locate in the crowded centers.
Wherever industry and commerce thrive, there cities will be built and
will grow. Half the population of the United States is now living in
towns and cities of over 2500 population. If the drift to the cities
continues at its present rate of progress, ninety per cent of the
people will be living in the cities and towns by 1980 and only ten per
cent in the country districts. That is already the case in England.[10]
One Englishman in every seven lives in London. That seems to be a
very striking fact until we remember that one American in every ten
lives in New York, Chicago, or Philadelphia. New York City has
nearly six millions now; in fifty years, at its present rate of growth, it
will be an urban giant of fifteen millions or more.
Why do the immigrants go to the cities rather Why most
than to the country districts? The chief reason is immigrants go to
that most immigrants come to the United States the cities.
to find work, and work is most easily found in the large cities. The
great majority of immigrants have learned no trade before they come
to America, hence they must seek jobs which require no great
amount of skill or training. Another reason is that the immigrants
want to be near others who speak their own language. In every large
city these foreign colonies or settlements are created—Italian,
Polish, Jewish, and so on. The newcomer naturally prefers to live in
one of these colonies until he learns the English language, but
having become accustomed to the city he rarely leaves it. Among the
many millions of immigrants who have come to America during the
past fifty years the great majority have gone to the cities, particularly
to the large cities. New York City, for example, contains today more
than two million people who were not born in this country.
Nevertheless, the cities have not been built up by the immigrant
alone. Large numbers of native Americans have left the rural districts
and have helped to swell the population of the urban centers.
The Ebb and Flow of Immigration.—The United States has been
the melting-pot of the nations. No other country has ever welcomed
to its shores so many millions of people from all parts of the earth.
One hundred years ago there were relatively few foreign-born
persons in the country. But waves of immigration soon began to
come and the number of incoming aliens, which was less than ten
thousand in 1790, rose to more than four hundred thousand in some
of the years preceding the Civil War. The immigrants during this
period were for the most part from England, Ireland, and Germany.
During the Civil War the influx subsided, but when the war was over
it quickly began to swell once more and it continued, with various
ups and downs, to the outbreak of the World War in 1914.
In some years, during this period since 1865 Where the
the number of immigrants has been as low as immigrants have
200,000; in other years it has run above a come from.
million. Nearly every European race has been represented in this
influx, although some have come in much larger numbers than
others. Down to about 1880 most of the newcomers were from
Ireland, England, Scotland, Germany, the Scandinavian countries,
and the other regions of Northern Europe. But since that date the
source of immigration has been steadily shifting. During the past
forty years a much larger proportion has been coming from Italy,
Austria, Hungary, Poland, Greece, and some of the smaller
countries.[11] This has brought in many millions of people who are not
of Celtic or Teutonic stock.
Upon the outbreak of the great European Effects of the war
conflict in 1914 the flow of immigration subsided on immigration.
quickly and while the war lasted it practically ceased altogether. This
was due in part to the reluctance of European countries to let their
people leave, and in part due to the lack of shipping, since all
available vessels were engaged in carrying troops, munitions, and
supplies. Immediately after the close of hostilities, however, the
exodus from Europe recommenced, and in the year 1920 the figures
of immigration once more rose into the hundreds of thousands. In
spite of the strict regulations imposed by the United States
government, enormous numbers of people in Poland, Italy, Hungary,
and various other countries began applying for permission to
migrate. The American consular offices in Europe were besieged by
long lines of people waiting all day for passports to the Promised
Land. In the face of this threatened flood Congress felt impelled to
place all immigration under strict limitation, as will be explained a few
paragraphs later.
During the hundred years from 1820 to 1920 it is estimated that
more than thirty million immigrants reached the United States, which
is the largest migration in history.[12] It is true that considerable
numbers of these immigrants eventually drifted back to their native
lands but the larger portion of them remained permanently here.
Causes of Immigration.—The causes of The various factors
immigration to this country are manifold. First in which have caused
importance among them is the economic Europeans to leave
attractiveness of the United States. Cheap land, their own countries.
equal opportunities, the ease with which any industrious man or
woman can make a living—these are the magnets which drew
millions of people across the Atlantic during the nineteenth century.
But there were other causes, namely, the existence in various parts
of Europe of conditions which encouraged the people to emigrate.
Religious persecution was one of the first things that drove men or
women to America. It brought the Pilgrims to Massachusetts in the
early days, the Quakers to Pennsylvania, and the English Catholics
to Maryland. Political oppression in the middle years of the
nineteenth century sent several million Germans to the United
States, particularly to the Middle West. Oppression, both religious
and political, has been instrumental in driving the Jews across the
seas. Overcrowding in both the cities and the rural regions of Europe
and the consequent inability of the people to obtain lands of their
own, has been responsible for much of the drift to America. The
exhaustion of the soil in Ireland, the crop failures and consequent
famines there, have led to the migrations of the Irish, although
dissatisfaction with political conditions has also been a factor in this
case. To a considerable extent, moreover, immigration has been
assisted by those aliens who were already settled in this country.
Immigrants arrived in this country and by hard work soon became
prosperous. Then they sent passage-money to their friends and
relatives until whole groups of families were enabled to come to
America in this way. The steamship companies, too, have advertised
the attractions of America in all parts of Europe; they have kept
agents at work in many countries, and have been an important factor
in promoting immigration.
Nevertheless the underlying causes of The economic
immigration are economic. Immigrants come in factors have been
greater numbers when prosperity reigns in the most important.
America than in periods of business depression. This indicates that
high wages and plenty of employment are the things which have the
greatest influence in bringing them here. The democratic institutions
of America have played their part, no doubt, and the relative social
equality which exists among the people of the United States has
been an attraction to those who are denied such equality in their own
homelands. But the equality of economic opportunity, the open
career, the chance of making a good living and even a fortune—
these, after all, have been and still are the impelling forces.
The Effects of Immigration.—A large How the immigrant
immigration is certain to have far-reaching has affected
effects, particularly if the newcomers be of a American life.
widely different race. It has assuredly had far-reaching effects in the
United States. Take the importation of the negroes as the most
conspicuous example. The coming of several hundred thousand
persons of African blood, during the years preceding 1808, has
resulted in placing a colored population of more than ten millions in
the heart of a white man’s country, with all the social, economic, and
political problems that this situation implies. It influenced the
agricultural system of the Southern states, provided the underlying
causes of the Civil War, and has profoundly affected the politics of
the United States down to the present day. The problem presented
by the immigration of the various European races has been
altogether different because these races can be assimilated whereas
the negroes can not. Even so, the European immigration has