Details

<p>Preface to the Third Edition xv</p> <p>List of Abbreviations and Symbols xvii</p> <p><b>1 General Aspects of Enzyme Analysis </b><b>1</b></p> <p>1.1 Introduction and Essentials for Enzyme Assays 1</p> <p>Standard Books, Series 4</p> <p>Databases 4</p> <p>1.2 Theoretical Basis of Enzyme Assays 4</p> <p>1.2.1 Order of Enzyme Reactions 4</p> <p>1.2.2 Importance of the Reaction Order for Enzyme Reactions 6</p> <p>1.2.3 The Reaction Velocity, Significance, and Practical Aspects 10</p> <p>1.2.3.1 Determination of the Reaction Velocity, the Progress Curve 10</p> <p>1.2.3.2 Enzyme Units 15</p> <p>1.2.3.3 A Short Discussion About Errors in Enzyme Assays 17</p> <p>1.2.3.4 Practical Rules for the Preparation of Dilution Series 21</p> <p>1.2.3.5 Statistical Treatment of Enzyme Reactions 23</p> <p>1.2.4 Treatment of the Michaelis–Menten Equation 24</p> <p>1.2.4.1 General Considerations 24</p> <p>1.2.4.2 Linear Representations of the Michaelis–Menten Equation 26</p> <p>1.2.5 Enzyme Inhibition 28</p> <p>1.2.6 Multisubstrate Reactions 33</p> <p>1.3 Essential Conditions for Enzyme Assays 35</p> <p>1.3.1 Dependence on Solvents and Ionic Strength 35</p> <p>1.3.2 pH Dependency 36</p> <p>1.3.2.1 Isoelectric Point 38</p> <p>1.3.2.2 Buffers: What Must Be Regarded? 38</p> <p>1.3.2.3 How to Prepare Buffers? 41</p> <p>1.3.3 Temperature Dependency 42</p> <p>1.3.4 Stability of Enzymes 47</p> <p>1.3.4.1 Why Are Enzymes Unstable? 47</p> <p>1.3.4.2 How Can Enzymes Be Stabilized? 48</p> <p>1.3.4.3 How to Store Enzymes? 48</p> <p>1.4 Theory of Coupled Enzyme Reactions 51</p> <p>1.4.1 Two Coupled Reactions 51</p> <p>1.4.2 Three Coupled Reactions 54</p> <p>1.5 Substrate Determination 54</p> <p>1.5.1 End Point Method 55</p> <p>1.5.2 Substrate Determination by Coupled Enzyme Reactions 56</p> <p>1.5.3 Kinetic Method for Substrate Determination 57</p> <p>1.5.4 Enzymatic Cycling 57</p> <p><b>2 Instrumental Aspects </b><b>61</b></p> <p>2.1 Spectroscopic Methods 61</p> <p>2.1.1 Absorption (UV/Vis) Photometry 61</p> <p>2.1.2 Cuvettes 72</p> <p>2.1.2.1 Shape 72</p> <p>2.1.2.2 Material 73</p> <p>2.1.2.3 Cleaning 73</p> <p>2.1.3 Turbidity Measurement 74</p> <p>2.1.4 Fluorescence Photometry 75</p> <p>2.1.5 Luminometry 79</p> <p>2.1.6 Polarimetry 80</p> <p>2.2 Electrochemical Methods 81</p> <p>2.2.1 pH Meter and Glass Electrodes 81</p> <p>2.2.2 pH Stat 82</p> <p>2.2.3 Potentiometry 83</p> <p>2.2.4 Oxygen and Carbon Dioxide Electrodes 83</p> <p>2.3 Radioactive Labeling 84</p> <p>2.4 Diverse Methods 84</p> <p><b>3 Enzyme Assays </b><b>87</b></p> <p>3.1 Enzyme Nomenclature 88</p> <p>3.2 Practical Considerations for Enzyme Assays 90</p> <p><b>4 Oxidoreductases, EC 1 </b><b>95</b></p> <p>4.1 General Assay Procedures 95</p> <p>4.1.1 Optical Assay 95</p> <p>4.1.2 Fluorimetric Assay 96</p> <p>4.2 Alcohol Dehydrogenase (ADH), EC 1.1.1.1 96</p> <p>4.2.1 Reduction Assay 96</p> <p>4.2.2 Oxidation Assay 97</p> <p>4.3 Alcohol Dehydrogenase (NADP⁺), EC 1.1.1.2 98</p> <p>4.4 Homoserine Dehydrogenase, EC 1.1.1.3 99</p> <p>4.5 Shikimate Dehydrogenase, EC 1.1.1.25 100</p> <p>4.6 l-Lactate Dehydrogenase (LDH), EC 1.1.1.27 101</p> <p>4.6.1 Photometric Reduction Assay 101</p> <p>4.6.2 Fluorimetric Reduction Assay 102</p> <p>4.6.3 Oxidation Assay 103</p> <p>4.7 Malate Dehydrogenase (MDH), EC 1.1.1.37 104</p> <p>4.8 Malate Dehydrogenase (Oxaloacetate-Decarboxylating) (NAD⁺), EC 1.1.1.38, and Malate Dehydrogenase (Decarboxylating), EC 1.1.1.39 105</p> <p>4.9 Malate Dehydrogenase (Oxaloacetate-decarboxylating) (NADP⁺), EC 1.1.1.40 106</p> <p>4.10 Isocitrate Dehydrogenase (NAD⁺) (ICDH), EC 1.1.1.41 107</p> <p>4.11 Isocitrate Dehydrogenase (NADP⁺) (ICDH), EC 1.1.1.42 108</p> <p>4.12 Glucose-6-Phosphate Dehydrogenase (NADP⁺), EC 1.1.1.49 (G6PDH) 109</p> <p>4.13 Glucose Oxidase (GOD), EC 1.1.3.4 110</p> <p>4.14 Formate Dehydrogenase (FDH), EC 1.2.1.2 111</p> <p>4.15 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), EC 1.2.1.12 112</p> <p>4.15.1 Oxidation Assay 112</p> <p>4.15.2 Reduction Assay Coupled with 3-Phosphoglycerate Kinase (PGK) 113</p> <p>4.16 Long-Chain-Aldehyde Dehydrogenase, EC 1.2.1.48 114</p> <p>4.17 Pyruvate Dehydrogenase (Acetyl-transferring) (PDH), EC 1.2.4.1 116</p> <p>4.17.1 Ferricyanide as Electron Acceptor 116</p> <p>4.17.2 Dichlorophenolindophenol as Electron Acceptor 117</p> <p>4.18 Aldehyde Oxidase, EC 1.2.3.1 118</p> <p>4.19 Oxoglutarate Dehydrogenase (Succinyl-transferring) (OGDH), EC 1.2.4.2 119</p> <p>4.20 Pyruvate Ferredoxin Oxidoreductase, EC 1.2.7.1 120</p> <p>4.20.1 Assay with Cytochrome c (cyt c) as Electron Acceptor 121</p> <p>4.21 Alanine Dehydrogenase, EC 1.4.1.1 121</p> <p>4.21.1 Oxidation of Alanine 122</p> <p>4.21.2 Reduction of Pyruvate 122</p> <p>4.22 Glutamate Dehydrogenase, EC 1.4.1.3 123</p> <p>4.23 Leucine Dehydrogenase, EC 1.4.1.9 124</p> <p>4.24 l-Amino-Acid Oxidase, EC 1.4.3.2 125</p> <p>4.25 d-Amino-Acid Oxidase, EC 1.4.3.3 126</p> <p>4.26 Monoamine Oxidase, EC 1.4.3.4 126</p> <p>4.27 Primary Amine Oxidase, EC 1.4.3.21 127</p> <p>4.27.1 Spectrophotometric Assay 128</p> <p>4.27.2 Polarographic Assay of O₂ Uptake with O₂ Electrode 128</p> <p>4.27.3 Assays for Benzylamine Oxidase Activity 129</p> <p>4.28 Diamine Oxidase, EC 1.4.3.22 129</p> <p>4.29 NADH:Ubiquinone Reductase (H⁺-Translocating) EC 1.6.5.3 130</p> <p>4.29.1 Spectrophotometric Assay 131</p> <p>4.30 NADH Dehydrogenase, EC 1.6.99.3 131</p> <p>4.31 Factor-Independent Urate Hydroxylase, EC 1.7.3.3 132</p> <p>4.32 Dihydrolipoyl Dehydrogenase, EC 1.8.1.4 133</p> <p>4.32.1 Oxidation of Dihydrolipoamide 134</p> <p>4.32.2 Reduction of Lipoamide 134</p> <p>4.33 Glutathione Disulfide Reductase, EC 1.8.1.7 135</p> <p>4.34 Cytochrome-c Oxidase (COX), EC 1.9.3.1 137</p> <p>4.34.1 Spectrophotometric Assay 137</p> <p>4.34.2 Assay with Oxygen Electrode 137</p> <p>4.35 Catalase, EC 1.11.1.6 138</p> <p>4.36 Peroxidase (POD) EC 1.11.1.7 139</p> <p>4.36.1 Assay with 2,2′-Azino-bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) 140</p> <p>4.36.2 Assay with Guaiacol 140</p> <p>4.36.3 Assay with Dianisidine 141</p> <p>4.37 Glutathione Peroxidase, EC 1.11.1.9 142</p> <p>4.37.1 Coupled Assay with Glutathione Reductase 142</p> <p>4.38 Photinus luciferin 4-Monooxigenase (ATP-Hydrolyzing), EC 1.13.12.7 143</p> <p>4.39 Alkylglycerol Monooxygenase, EC 1.14.16.5 145</p> <p>4.39.1 Spectroscopic Assay 145</p> <p>4.39.2 Coupled Assay with HPLC Detection 145</p> <p>4.40 Dopamine ß-Monooxygenase, EC 1.14.17.1 147</p> <p>4.41 Tyrosinase, EC 1.14.18.1 148</p> <p>4.41.1 Dopa Oxidase Assay 148</p> <p>4.41.2 Dopachrome Assay 149</p> <p>4.42 Superoxide Dismutase (SOD), EC 1.15.1.1 149</p> <p>4.42.1 Assay with Pyrogallol 150</p> <p>4.42.2 Assay with Ferricytochrome c and Xanthine Oxidase 150</p> <p>4.43 Xanthine Oxidase (XOD), EC 1.17.3.2 151</p> <p><b>5 Transferases, EC 2 </b><b>153</b></p> <p>5.1 Ornithine Carbamoyltransferase (OTC), EC 2.1.3.3 153</p> <p>5.1.1 Method 1 for Color Development 154</p> <p>5.1.2 Method 2 for Color Development 154</p> <p>5.2 Choline <i>O</i>-Acetyltransferase, EC 2.3.1.6 155</p> <p>5.3 Carnitine <i>O</i>-acetyltransferase, EC 2.3.1.7 156</p> <p>5.3.1 Direct Spectroscopic Assay 156</p> <p>5.3.2 Assay with DTNB 157</p> <p>5.4 Dihydrolipoamide Acetyltransferase, EC 2.3.1.12 157</p> <p>5.4.1 Spectrophotometric Assay 158</p> <p>5.4.2 Stopped Assay 159</p> <p>5.5 Fatty Acid Synthase, EC 2.3.1.85 160</p> <p>5.6 𝛾-Glutamyltransferase, EC 2.3.2.2 161</p> <p>5.7 Citrate Synthases, EC 2.3.3.1, EC 2.3.3.3, and EC 2.3.3.16 162</p> <p>5.8 ATP Citrate Lyase, EC 2.3.3.8 164</p> <p>5.9 Glycogen Phosphorylase, EC 2.4.1.1 165</p> <p>5.10 Purine-nucleoside Phosphorylase (PNP), EC 2.4.2.1 166</p> <p>5.11 Glutathione Transferase, EC 2.5.1.18 167</p> <p>5.11.1 Spectrophotometric Assay 168</p> <p>5.11.2 Titrimetric Assay 168</p> <p>5.12 Aspartate Transaminase (AAT), EC 2.6.1.1 169</p> <p>5.13 Alanine Transaminase, EC 2.6.1.2 170</p> <p>5.14 Tyrosine Transaminase (TAT), EC 2.6.1.5; Tryptophan Transaminase (Tam 1), EC 2.6.1.27; Phenylalanine (Histidine) Transaminase, EC 2.6.1.58 171</p> <p>5.14.1 Tyrosine Transaminase 171</p> <p>5.14.2 Tryptophan Transaminase 171</p> <p>5.14.3 Phenylalanine (Histidine) Transaminase: 171</p> <p>5.15 Hexokinase (HK), EC 2.7.1.1, Glucokinase (GK), EC 2.7.1.2 173</p> <p>5.16 Pyruvate Kinase (PK), EC 2.7.1.40 174</p> <p>5.17 Acetate Kinase, EC 2.7.2.1 176</p> <p>5.18 Phosphoglycerate Kinase (PGK), EC 2.7.2.3 177</p> <p>5.19 Aspartate Kinase (AK), EC 2.7.2.4 178</p> <p>5.20 Creatine Kinase (CK), EC 2.7.3.2 180</p> <p>5.20.1 Coupled Assay 180</p> <p>5.20.2 pH-Colorimetric Assay 181</p> <p><b>6 Hydrolases, EC 3 </b><b>183</b></p> <p>6.1 Triacylglycerol Lipase, EC 3.1.1.3 183</p> <p>6.1.1 Assay with pH Stat (Auto-titrator) 183</p> <p>6.1.2 Fluorimetric Assay 184</p> <p>6.2 Phospholipase A₂, EC 3.1.1.4 185</p> <p>6.3 Acetylcholinesterase (AChE), EC 3.1.1.7 186</p> <p>6.4 Cholinesterase (ButChE), EC 3.1.1.8 187</p> <p>6.4.1 pH Stat Assay 187</p> <p>6.4.2 Colorimetric Assay 188</p> <p>6.5 Hydroxyacylglutathione Hydrolase, EC 3.1.2.6 189</p> <p>6.5.1 Direct Assay 189</p> <p>6.5.2 Assay with DTNB 189</p> <p>6.6 <i>S</i>-Formylglutathione Hydrolase, EC 3.1.2.12 190</p> <p>6.7 Alkaline Phosphatase, EC 3.1.3.1 191</p> <p>6.7.1 Mammalian Alkaline Phosphatase 191</p> <p>6.7.2 Bacterial Alkaline Phosphatase 192</p> <p>6.8 Acid Phosphatase, EC 3.1.3.2 192</p> <p>6.9 5′-Nucleotidase, EC 3.1.3.5 193</p> <p>6.9.1 Assay by Determination of P_i 194</p> <p>6.9.2 Assay by Converting Adenosine into Inosine 194</p> <p>6.10 Glucose-6-Phosphatase, EC 3.1.3.9 195</p> <p>6.11 3′,5′-Cyclic-Nucleotide Phosphodiesterase, EC 3.1.4.17 196</p> <p>6.12 Steryl-Sulfatase, EC 3.1.6.2 198</p> <p>6.13 Pancreatic Ribonuclease, EC 3.1.27.5 198</p> <p>6.14 α-Amylase, EC 3.2.1.1 199</p> <p>6.15 Glucan 1,4-α-Glucosidase (AMG), EC 3.2.1.3 201</p> <p>6.15.1 Coupled Assay with HK and G6PDH 201</p> <p>6.15.2 Photometric Assay with 4-Nitrophenyl-d-Glucose 202</p> <p>6.15.3 Fluorimetric Assay with 4-Methylumbelliferyl-α-d-Glucoside 202</p> <p>6.16 Cellulases 203</p> <p>6.16.1 β-1,4-Glucanase, EC 3.2.1.4 203</p> <p>6.16.2 β-Glucosidase, EC 3.2.1.21 203</p> <p>6.16.3 Orcinol Assay 204</p> <p>6.16.4 Activity Staining 204</p> <p>6.17 Lysozym, EC 3.2.1.17 206</p> <p>6.18 Sialidase, EC 3.2.1.18 207</p> <p>6.18.1 Fluorimetric Assay 207</p> <p>6.18.2 Activity Staining 208</p> <p>6.19 α-Glucosidase, EC 3.2.1.20 208</p> <p>6.19.1 Coupled Assay 208</p> <p>6.19.1.1 α-Glucosidase Reaction 209</p> <p>6.19.1.2 Glucose Determination 209</p> <p>6.19.2 Assay with 4-Nitrophenylglucopyranoside 210</p> <p>6.20 β-Galactosidase, EC 3.2.1.23 211</p> <p>6.21 α-Mannosidase, EC 3.2.1.24 212</p> <p>6.21.1 Photometric Microassay 212</p> <p>6.21.2 Fluorimetric Assay 213</p> <p>6.22 β-Fructofuranosidase, EC 3.2.1.26 213</p> <p>6.23 β-Glucuronidase, EC 3.2.1.31 214</p> <p>6.23.1 Fluorimetric Assay 215</p> <p>6.24 β-<i>N</i>-Acetylhexosaminidase, EC 3.2.1.52 215</p> <p>6.25 Proteases, EC 3.4, General Assays 216</p> <p>6.25.1 Anson Assay 216</p> <p>6.25.2 Casein Assay 218</p> <p>6.25.3 Azocasein Assay 219</p> <p>6.25.4 Ninhydrin Assay 220</p> <p>6.26 Leucyl Aminopeptidase (LAP), EC 3.4.11.1, Bacterial Leucyl Aminopeptidase, EC 3.4.11.10 221</p> <p>6.26.1 Assay with Leucineamide 222</p> <p>6.26.2 Assay with Leucine-<i>p</i>-nitroanilide 222</p> <p>6.27 Peptidyl-dipeptidase A, EC 3.4.15.1 223</p> <p>6.28 α-Chymotrypsin, EC 3.4.21.1 224</p> <p>6.28.1 Assay with SUPHEPA 224</p> <p>6.28.2 Assay with GLUPHEPA 225</p> <p>6.29 Trypsin, EC 3.4.21.4 226</p> <p>6.30 Pancreatic Elastase, EC 3.4.21.35 227</p> <p>6.30.1 Assay with Succinyl-Ala–Ala–Ala–<i>p</i>-Nitroanilide 227</p> <p>6.30.2 Esterase Activity of Elastase 227</p> <p>6.31 Cathepsin B, EC 3.4.22.1 228</p> <p>6.32 Pepsin A, EC 3.4.23.1 229</p> <p>6.33 Asparaginase, EC 3.5.1.1 230</p> <p>6.34 Glutaminase, EC 3.5.1.2 232</p> <p>6.34.1 Determination of Ammonia with Nessler’s Reagent 232</p> <p>6.34.2 pH Stat Assay 233</p> <p>6.35 Urease, EC 3.5.1.5 233</p> <p>6.35.1 pH Stat Assay 234</p> <p>6.35.2 Photometric Assay 234</p> <p>6.36 Guanine Deaminase, EC 3.5.4.3 235</p> <p>6.36.1 Determination of Ammonia 236</p> <p>6.37 Adenosinetriphosphatase, EC 3.6.1.3 237</p> <p>6.38 Mg²⁺ Importing ATPase, EC 3.6.3.2, Na⁺/K⁺-Exchanging ATPase, EC 3.6.3.9 238</p> <p>6.38.1 Assay of Total ATPase Activity 238</p> <p>6.38.2 Assay of Mg²⁺-ATPase Activity 239</p> <p><b>7 Lyases, EC 4 </b><b>241</b></p> <p>7.1 Pyruvate Decarboxylase (PDC), EC 4.1.1.1 241</p> <p>7.2 Glutamate Decarboxylase (GAD), EC 4.1.1.15 242</p> <p>7.3 Fructose-bisphosphate Aldolase, EC 4.1.2.13 244</p> <p>7.4 Anthranilate Synthase, EC 4.1.3.27 245</p> <p>7.5 Carbonic Anhydrase (CA), EC 4.2.1.1 246</p> <p>7.5.1 pH Stat Assay 246</p> <p>7.5.2 Esterase Assay with 4-Nitrophenylacetate 247</p> <p>7.6 Fumarate Hydratase, EC 4.2.1.2 248</p> <p>7.7 Lactoylglutathione Lyase, EC 4.4.1.5 249</p> <p>7.8 Adenylate Cyclase (AC), EC 4.6.1.1 250</p> <p><b>8 Isomerases, EC 5 </b><b>253</b></p> <p>8.1 Xylose Isomerase, EC 5.3.1.5 253</p> <p>8.1.1 D-Xylose Isomerase Assay 253</p> <p>8.1.2 D-Xylose Isomerase Microplate Assay 254</p> <p>8.1.3 D-Glucose Isomerase Assay 255</p> <p>8.1.4 D-Glucose Isomerase Microplate Assay 256</p> <p>8.2 Glucose-6-phosphate Isomerase (G6PI), EC 5.3.1.9 256</p> <p>8.3 Phosphoglucomutase (PGM), EC 5.4.2.2 257</p> <p><b>9 Ligases (Synthetases), EC 6 </b><b>261</b></p> <p>9.1 Tyrosine-tRNA Ligase, EC 6.1.1.1 261</p> <p>9.1.1 Fluorimetric Assay 261</p> <p>9.1.2 ATP–³²PP Exchange 262</p> <p>9.2 Acetate-CoA Ligase (ACL), EC 6.2.1.1 263</p> <p>9.2.1 Direct Radioactive Assay 264</p> <p>9.2.2 Coupled Spectroscopic Assay 264</p> <p>9.3 Glutamine Synthetase, EC 6.3.1.2 266</p> <p><b>10 Assays for Multi-enzyme Complexes </b><b>269</b></p> <p>10.1 Pyruvate Dehydrogenase Complex (PDHC) 269</p> <p>10.1.1 Overall Activity of PDHC by NAD⁺ Reduction 270</p> <p>10.1.2 Overall Activity of PDHC by Dismutation Assay 270</p> <p>10.2 α-Oxoglutarate Dehydrogenase Complex (OGDHC) 272</p> <p>10.2.1 Overall Activity by NAD⁺ Reduction 273</p> <p><b>11 Assays for Other Enzyme Relevant Parameters </b><b>275</b></p> <p>11.1 Substrate Determination 275</p> <p>11.1.1 Determination of NADP(H) by Enzymatic Cycling 275</p> <p>11.1.1.1 Cycling Reaction 276</p> <p>11.1.2 Determination of NAD(H) by Enzymatic Cycling 277</p> <p>11.2 Protein Determination 279</p> <p>11.2.1 Biuret Assay 279</p> <p>11.2.2 BCA Assay 281</p> <p>11.2.2.1 Assay for Soluble Proteins 281</p> <p>11.2.2.2 Modification for Immobilized Proteins 282</p> <p>11.2.3 Lowry Assay 282</p> <p>11.2.4 Coomassie Binding Assay (Bradford Assay) 283</p> <p>11.2.4.1 Assay for Soluble Proteins 284</p> <p>11.2.4.2 Modification for Immobilized Proteins 284</p> <p>11.2.5 Absorption Method 285</p> <p>11.2.6 Fluorimetric Assay 287</p> <p>11.2.7 Ninhydrin Assay 288</p> <p>11.2.7.1 Ninhydrin Assay with Hydrolysis 288</p> <p>11.2.7.2 Modified Ninhydrin AssayWithout Hydrolysis 289</p> <p>11.2.8 Protein Assay with 2-Hydroxy-1-naphthaldehyde 290 General Literature for Protein Assays 291</p> <p>11.3 Phosphate Determination 291</p> <p>11.4 Determination of Metal Ions 293</p> <p>11.4.1 Calcium and Magnesium 293</p> <p>11.4.2 Iron 294</p> <p>11.4.2.1 Determination with Ferrozine 295</p> <p>11.4.2.2 Determination of Fe^II with 1,10-Phenanthroline in the Presence of Fe^III 295</p> <p>11.4.3 Copper 296</p> <p>11.4.3.1 Biquinoline Method 296</p> <p>11.4.3.2 Oxalyldihydrazide Method 297</p> <p>11.4.4 Manganese 297</p> <p>11.4.4.1 Colorimetric Assay 298</p> <p>11.4.4.2 Assay with 1-(2-Pyridylazo)-2-naphthol (PAN) 298</p> <p>11.4.5 Zinc 299</p> <p>11.5 Glycoprotein Assays 300</p> <p>11.5.1 Identification in Electrophoresis Gels 300</p> <p>11.5.2 Quantitative Analysis of Protein-Bound Hexoses 300</p> <p>11.6 Cross-linking of Proteins with Dimethylsuberimidate 301</p> <p>11.7 Concentrating Enzyme Solutions 302</p> <p>11.7.1 Precipitation 303</p> <p>11.7.2 Ultrafiltration and Dialysis 306</p> <p>11.7.3 Ultracentrifugation 307</p> <p>11.7.4 Lyophilization 307</p> <p>11.7.5 Other Concentration Methods 307</p> <p><b>12 Enzyme Immunoassays </b><b>309</b></p> <p>12.1 Radioimmunoassays 309</p> <p>12.2 Principle of Enzyme Immunoassays 309</p> <p>12.3 Noncompetitive Solid-Phase Enzyme Immunoassay 311</p> <p>12.4 Competitive Solid-Phase Enzyme Immunoassay 312</p> <p>12.5 Methods for Enzyme Immunoassays and Immobilization Techniques 312</p> <p>12.5.1 Protein Coupling to Cyanogen Bromide Activated Agarose 312</p> <p>12.5.2 Coupling of Diaminohexyl Spacer 313</p> <p>12.5.3 Periodate Activation of Cellulose 314</p> <p>12.5.4 Introduction of Thiol Groups into Proteins (Antibodies) 315</p> <p>12.5.5 Conjugation of a Protein (Antibody) with an Enzyme (Peroxidase) 316</p> <p>12.5.6 Conjugation of ß-Galactosidase to Proteins (Antibodies) by MBS 316</p> <p>12.5.7 Conjugation of Alkaline Phosphatase to Antibodies by Glutaraldehyde 317</p> <p><b>13 Binding Measurements </b><b>319</b></p> <p>13.1 Different Types of Binding 319</p> <p>13.1.1 General Considerations 319</p> <p>13.1.2 How Can Specific Reversible Binding be Identified? 320</p> <p>13.1.3 Experimental Aspects 322</p> <p>13.2 Binding Measurements by Size Discrimination 325</p> <p>13.2.1 Equilibrium Dialysis 325</p> <p>13.2.1.1 Binding of Indole to Bovine Serum Albumin 327</p> <p>13.2.2 Evaluation of Binding Experiments 329</p> <p>13.2.3 Ultrafiltration 330</p> <p>13.2.4 Gel Filtration 331</p> <p>13.2.5 Ultracentrifugation 332</p> <p>13.3 Spectroscopic Methods 333</p> <p>13.3.1 Difference Spectroscopy 334</p> <p>13.3.1.1 Difference Spectroscopic Titration of Ligands Binding to Catalase 336</p> <p>13.3.1.2 Evaluation of Spectroscopic Binding Curves 339</p> <p>13.3.2 Fluorescence Spectroscopy 341</p> <p>13.3.2.1 Binding of ANS to Bovine Serum Albumin 341</p> <p>13.4 Other Binding Methods 344</p> <p>13.4.1 Radioactive Labeling 344</p> <p>13.4.2 Surface Plasmon Resonance (SPR) 345</p> <p><b>14 Enzymes in Technical Applications </b><b>347</b></p> <p>14.1 Modes of Enzyme Immobilization 347</p> <p>14.1.1 Adsorption 348</p> <p>14.1.2 Entrapment 350</p> <p>14.1.3 Encapsulation 350</p> <p>14.1.4 Cross-linking 351</p> <p>14.1.5 Covalent Immobilization to Solid Supports 351</p> <p>14.1.5.1 Supports 351</p> <p>14.1.5.2 Spacer 353</p> <p>14.2 Methods for Enzyme Immobilization 354</p> <p>14.2.1 Microencapsulation in Nylon Beads 355</p> <p>14.2.2 Entrapment in Polyacrylamide 355</p> <p>14.2.3 Covalent Immobilization on Glass Surfaces 356</p> <p>14.2.4 Covalent Immobilization on Controlled-Pore Glass (CPG) 358</p> <p>14.2.5 Covalent Immobilization to Polyamide 360</p> <p>14.2.5.1 O-Alkylation with Triethyloxonium Tetrafluoroborate 361</p> <p>14.2.5.2 Immobilization to Amino Groups after Partial Hydrolysis of Polyamide 363</p> <p>14.2.5.3 Immobilization to Carboxyl Groups After Partial Hydrolysis of Polyamide 364</p> <p>14.2.6 Immobilization to Polyester 365</p> <p>14.2.7 Immobilization by Alkaline Hydrolysis and Activation with Tosylchloride 367</p> <p>14.2.8 Alkaline Hydrolysis and Activation by Carbonyldiimidazol 368</p> <p>14.3 Analysis of Immobilized Enzymes 368</p> <p>14.3.1 General Principles 368</p> <p>14.3.2 Continuous Photometric Assays for Immobilized Enzymes 369</p> <p>14.3.3 Cofactors in Reactions with Immobilized Enzymes 371</p> <p>14.4 Enzyme Reactors 372</p> <p>14.4.1 Batch Reactor (Stirred-Tank Reactor) 373</p> <p>14.4.2 Membrane Reactor 373</p> <p>14.4.3 Solid-Bed Reactor 374</p> <p>14.4.4 Immobilized Cells 375</p> <p>14.5 Biosensors 375</p> <p>14.5.1 Enzyme Electrodes 375</p> <p>14.5.2 Immunoelectrodes 379</p> <p>14.5.3 Other Biosensors 379</p> <p>14.6 Immobilized Enzymes in Therapy 381</p> <p>Index 383</p>

Hans Bisswanger was Professor at the Interfaculty Institute of Biochemistry at the University of Tübingen (Germany), where he has developed and taught for many years an intensive course on enzyme kinetics, enzyme technology and ligand binding. His scientific interest lies with structural and regulatory mechanisms of multi-enzyme complexes, thermophilic enzymes and the technical application of immobilized enzymes. He is the author of several well-known books on enzymology that have appeared in different languages and editions.

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