Bioanalytical Chemistry - S. Mikkelsen, E. Corton (Wiley, 2004) WW

377 Pages·2004·4.53 MB·English

BIOANALYTICAL
CHEMISTRY

Susan R. Mikkelsen
Eduardo Corto´n

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright # 2004 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.
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Library of Congress Cataloging-in-Publication Data:

Mikkelsen, Susan R., 1960–
Bioanalytical chemistry
/
Susan R. Mikkelsen, Eduardo Corto´n.
p.
cm.
Includes bibliographical references and index.
ISBN 0-471-54447-7
(cloth)
1. Analytical biochemistry.
[DNLM:
1. Chemistry, Analytical.
QY 90 M637b 2004]
I. Corto´n,
Eduardo, 1962– II. Title.
QP519.7.M54 2004
5720.36–dc22
2003016568

Printed in the United States of America

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1

CONTENTS

Preface
xv

Acknowledgments
xvii

1.
Spectroscopic Methods for Matrix Characterization
1

1.1
Introduction
1
1.2
Total Protein
2
1.2.1
Lowry Method
3
1.2.2
Smith (BCA) Method
3
1.2.3
Bradford Method
4
1.2.4
Ninhydrin-Based Assay
5
1.2.5
Other Protein Quantitation Methods
5
1.3
Total DNA
7
1.3.1
Diaminobenzoic Acid Method
7
1.3.2
Diphenylamine Method
9
1.3.3
Other Fluorometric Methods
9
1.4
Total RNA
10
1.5
Total Carbohydrate
11
1.5.1
Ferricyanide Method
11
1.5.2
Phenol–Sulfuric Acid Method
11
1.5.3
2-Aminothiophenol Method
12
1.5.4
Purpald Assay for Bacterial Polysaccharides
12
1.6
Free Fatty Acids
13
References
14
Problems
15

2.
Enzymes
16

2.1
Introduction
16
2.2
Enzyme Nomenclature
17
2.3
Enzyme Commission Numbers
18
2.4
Enzymes in Bioanalytical Chemistry
19
2.5
Enzyme Kinetics
21
2.5.1
Simple One-Substrate Enzyme Kinetics
23
2.5.2
Experimental Determination of Michaelis–Menten
Parameters
24

v

2.5.2.1 Eadie–Hofstee Method
25
2.5.2.2 Hanes Method
25
2.5.2.3 Lineweaver–Burk Method
26
2.5.2.4 Cornish–Bowden–Eisenthal Method
27
2.5.3
Comparison of Methods for the Determination
of Km Values
28
2.5.4
One-Substrate, Two-Product Enzyme Kinetics
29
2.5.5
Two-Substrate Enzyme Kinetics
29
2.5.6
Examples of Enzyme-Catalyzed Reactions and
Their Treatment
31
2.6
Enzyme Activators
32
2.7
Enzyme Inhibitors
33
2.7.1
Competitive Inhibition
34
2.7.2
Noncompetitive Inhibition
35
2.7.3
Uncompetitive Inhibition
35
2.8
Enzyme Units and Concentrations
36
Suggested References
38
References
38
Problems
38

3.
Quantitation of Enzymes and Their Substrates
41

3.1
Introduction
41
3.2
Substrate Depletion or Product Accumulation
42
3.3
Direct and Coupled Measurements
43
3.4
Classification of Methods
45
3.5
Instrumental Methods
47
3.5.1
Optical Detection
47
3.5.1.1 Absorbance
47
3.5.1.2 Fluorescence
49
3.5.1.3 Luminescence
51
3.5.1.4 Nephelometry
53
3.5.2
Electrochemical Detection
53
3.5.2.1 Amperometry
53
3.5.2.2 Potentiometry
54
3.5.2.3 Conductimetry
54
3.5.3
Other Detection Methods
55
3.5.3.1 Radiochemical
55
3.5.3.2 Manometry
55
3.5.3.3 Calorimetry
56
3.6
Ultra-High-Throughput Assays (HTA)
56
3.7
Practical Considerations for Enzymatic Assays
57
Suggested References
57

vi
CONTENTS

References
57
Problems
58

4.
Immobilized Enzymes
61

4.1
Introduction
61
4.2
Immobilization Methods
61
4.2.1
Nonpolymerizing Covalent Immobilization
62
4.2.1.1 Controlled-Pore Glass
63
4.2.1.2 Polysaccharides
64
4.2.1.3 Polyacrylamide
65
4.2.1.4 Acidic Supports
66
4.2.1.5 Anhydride Groups
67
4.2.1.6 Thiol Groups
67
4.2.2
Cross-Linking with Bifunctional Reagents
68
4.2.3
Adsorption
69
4.2.4
Entrapment
69
4.2.5
Microencapsulation
70
4.3
Properties of Immobilized Enzymes
71
4.4
Immobilized Enzyme Reactors
76
4.5
Theoretical Treatment of Packed-Bed Enzyme Reactors
79
Suggested References
82
References
82
Problems
83

5.
Antibodies
86

5.1
Introduction
86
5.2
Structural and Functional Properties of Antibodies
87
5.3
Polyclonal and Monoclonal Antibodies
90
5.4
Antibody–Antigen Interactions
91
5.5
Analytical Applications of Secondary Antibody–Antigen
Interactions
93
5.5.1
Agglutination Reactions
93
5.5.2
Precipitation Reactions
94
Suggested References
97
References
97
Problems
98

6.
Quantitative Immunoassays with Labels
99

6.1
Introduction
99
6.2
Labeling Reactions
101
6.3
Heterogeneous Immunoassays
102

CONTENTS
vii

6.3.1
Labeled-Antibody Methods
104
6.3.2
Labeled-Ligand Assays
104
6.3.3
Radioisotopes
106
6.3.4
Fluorophores
107
6.3.4.1 Indirect Fluorescence
108
6.3.4.2 Competitive Fluorescence
108
6.3.4.3 Sandwich Fluorescence
108
6.3.4.4 Fluorescence Excitation Transfer
108
6.3.4.5 Time-Resolved Fluorescence
109
6.3.5
Chemiluminescent Labels
110
6.3.6
Enzyme Labels
112
6.4
Homogeneous Immunoassays
116
6.4.1
Fluorescent Labels
116
6.4.1.1 Enhancement Fluorescence
116
6.4.1.2 Direct Quenching Fluorescence
116
6.4.1.3 Indirect Quenching Fluorescence
117
6.4.1.4 Fluorescence Polarization Immunoassay
117
6.4.1.5 Fluorescence Excitation Transfer
118
6.4.2
Enzyme Labels
118
6.4.2.1 Enzyme-Multiplied Immunoassay Technique
118
6.4.2.2 Substrate-Labeled Fluorescein Immunoassay
119
6.4.2.3 Apoenzyme Reactivation Immunoassay (ARIS)
119
6.4.2.4 Cloned Enzyme Donor Immunoassay
120
6.4.2.5 Enzyme Inhibitory Homogeneous Immunoassay
120
6.5
Evaluation of New Immunoassay Methods
121
Suggested References
126
References
126
Problems
127

7.
Biosensors
131

7.1
Introduction
131
7.2
Response of Enzyme-Based Biosensors
132
7.3
Examples of Biosensor Configurations
135
7.3.1
Ferrocene-Mediated Amperometric Glucose Sensor
135
7.3.2
Potentiometric Biosensor for Phenyl Acetate
137
7.3.3
Potentiometric Immunosensor for Digoxin
138
7.3.4
Evanescent-Wave Fluorescence Biosensor
for Bungarotoxin
139
7.3.5
Optical Biosensor for Glucose Based on
Fluorescence Energy Transfer
141
7.3.6
Piezoelectric Sensor for Nucleic Acid Detection
142
7.3.7
Enzyme Thermistors
144

viii
CONTENTS

7.4
Evaluation of Biosensor Performance
145
Suggested References
147
References
147
Problems
148

8.
Directed Evolution for the Design of Macromolecular
Bioassay Reagents
150

8.1
Introduction
150
8.2
Rational Design and Directed Evolution
152
8.3
Generation of Genetic Diversity
154
8.3.1
Polymerase Chain Reaction and Error-Prone PCR
155
8.3.2
DNA Shuffling
157
8.4
Linking Genotype and Phenotype
158
8.4.1
Cell Expression and Cell Surface Display (in vivo)
158
8.4.2
Phage Display (in vivo)
159
8.4.3
Ribosome Display (in vitro)
160
8.4.4
mRNA-Peptide Fusion (in vitro)
160
8.4.5
Microcompartmentalization (in vitro)
160
8.5
Identification and Selection of Successful Variants
161
8.5.1
Identification of Successful Variants Based on
Binding Properties
162
8.5.2
Identification of Successful Variants Based on
Catalytic Activity
163
8.6
Directed Evolution of Galactose Oxidase
164
Suggested References
165
References
165
Problems
166

9.
Principles of Electrophoresis
167

9.1
Introduction
167
9.2
Electrophoretic Support Media
171
9.2.1
Paper
171
9.2.2
Starch Gels
172
9.2.3
Polyacrylamide Gels
173
9.2.4
Agarose Gels
177
9.2.5
Polyacrylamide–Agarose Gels
177
9.3
Effect of Experimental Conditions on
Electrophoretic Separations
177
9.4
Electric Field Strength Gradients
178
9.5
Detection of Proteins and Nucleic Acids After
Electrophoretic Separation
180
9.5.1
Stains and Dyes
181

CONTENTS
ix

9.5.2
Detection of Enzymes by Substrate Staining
183
9.5.3
The Southern Blot
184
9.5.4
The Northern Blot
184
9.5.5
The Western Blot
185
9.5.6
Detection of DNA Fragments on Membranes
with DNA Probes
185
Suggested References
188
References
188
Problems
189

10.
Applications of Zone Electrophoresis
191

10.1
Introduction
191
10.2
Determination of Protein Net Charge and Molecular Weight
Using PAGE
191
10.3
Determination of Protein Subunit Composition and Subunit
Molecular Weights
193
10.4
Molecular Weight of DNA by Agarose Gel Electrophoresis
195
10.5
Identification of Isoenzymes
196
10.6
Diagnosis of Genetic (Inherited) Disease
197
10.7
DNA Fingerprinting and Restriction Fragment
Length Polymorphism
199
10.8
DNA Sequencing with the Maxam–Gilbert Method
202
10.9
Immunoelectrophoresis
206
Suggested References
210
References
211
Problems
211

11.
Isoelectric Focusing
213

11.1
Introduction
213
11.2
Carrier Ampholytes
214
11.3
Modern IEF with Carrier Ampholytes
216
11.4
Immobilized pH Gradients (IPGs)
219
11.5
Two-Dimensional Electrophoresis
222
Suggested References
224
References
225
Problems
225

12.
Capillary Electrophoresis
227

12.1
Introduction
227
12.2
Electroosmosis
229
12.3
Elution of Sample Components
229
12.4
Sample Introduction
230

x
CONTENTS

12.5
Detectors for Capillary Electrophoresis
231
12.5.1
Laser-Induced Fluorescence Detection
232
12.5.2
Mass Spectrometric Detection
235
12.5.3
Amperometric Detection
236
12.5.4
Radiochemical Detection
239
12.6
Capillary Polyacrylamide Gel Electrophoresis (C-PAGE)
240
12.7
Capillary Isoelectric Focusing (CIEF)
242
Suggested References
244
References
244
Problems
244

13.
Centrifugation Methods
247

13.1
Introduction
247
13.2
Sedimentation and Relative Centrifugal g Force
247
13.3
Centrifugal Forces in Different Rotor Types
249
13.3.1
Swinging-Bucket Rotors
249
13.3.2
Fixed-Angle Rotors
250
13.3.3
Vertical Rotors
250
13.4
Clearing Factor (k)
251
13.5
Density Gradients
252
13.5.1
Materials Used to Generate a Gradient
252
13.5.2
Constructing Pre-Formed and Self-Generated
Gradients
253
13.5.3
Redistribution of the Gradient in Fixed-Angle
and Vertical Rotors
254
13.6
Types of Centrifugation Techniques
255
13.6.1
Differential Centrifugation
255
13.6.2
Rate-Zonal Centrifugation
256
13.6.3
Isopycnic Centrifugation
257
13.7
Harvesting Samples
257
13.8
Analytical Ultracentrifugation
257
13.8.1
Instrumentation
258
13.8.2
Sedimentation Velocity Analysis
259
13.8.3
Sedimentation Equilibrium Analysis
262
13.9
Selected Examples
263
13.9.1
Analytical Ultracentrifugation for Quaternary
Structure Elucidation
263
13.9.2
Isolation of Retroviruses by Self-Generated Gradients
264
13.9.3
Isolation of Lipoproteins from Human Plasma
264
Suggested References
265
References
265
Problems
266

CONTENTS
xi

14.
Chromatography of Biomolecules
268

14.1
Introduction
268
14.2
Units and Definitions
268
14.3
Plate Theory of Chromatography
269
14.4
Rate Theory of Chromatography
270
14.5
Size Exclusion (Gel Filtration) Chromatography
272
14.6
Gel Matrices for Size Exclusion Chromatography
277
14.7
Affinity Chromatography
278
14.7.1
Immobilization of Affinity Ligands
280
14.7.2
Elution Methods
281
14.7.3
Determination of Association Constants by
High-Performance Affinity Chromatography
283
14.8
Ion-Exchange Chromatography
286
14.8.1
Retention Model for Ion-Exchange Chromatography
of Polyelectrolytes
288
Suggested References
292
References
293
Problems
293

15.
Mass Spectrometry of Biomolecules
295

15.1
Introduction
295
15.2
Basic Description of the Instrumentation
297
15.2.1
Soft Ionization Sources
297
15.2.1.1 Fast Atom–Ion Bombardment
297
15.2.1.2 Electrospray Ionization
299
15.2.1.3 Matrix-Assisted Laser
Desorption/Ionization
299
15.2.2
Mass Analyzers
300
15.2.3
Detectors
303
15.3
Interpretation of Mass Spectra
304
15.4
Biomolecule Molecular Weight Determination
308
15.5
Protein Identification
310
15.6
Protein–Peptide Sequencing
312
15.7
Nucleic Acid Applications
315
15.8
Bacterial Mass Spectrometry
318
Suggested References
318
References
319
Problems
320

16.
Validation of New Bioanalytical Methods
322

16.1
Introduction
322
16.2
Precision and Accuracy
323

xii
CONTENTS