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Section A:General1

Ⅰ．Principles of Enzymatic Analysis3

Ⅱ．Experimental Techniques14

Ⅲ．Cell and Tissue Disintegration43

Section B:Estimation of Substrates Preliminary Remarks57

Ⅰ．Substances Involved in Carbohydrate Metabolism59

1．Poly-,Oligo-and Disaccharides59

a)Glycogen59

b)Starch63

c)Cellulose64

d)Hemicelluloses72

e)Heparin79

Determination with ribonuclease79

Determination with pyruvic kinase84

f)Hyaluronic acid87

Spectrophotometric method87

Colorimetric method90

g)Raffinose93

h)Sucrose99

i)Lactose103

2．Monosaccharides and Derivatives107

a)D-Sedoheptulose-7-phosphate107

b)D-Sedoheptulose-1,7-diphosphate111

Determination with aldolase111

Determination with SDPase113

c)D-Glucose117

Determination with HK/G6P-DH117

Determination with GOD/POD123

d)D-Glucose-1-phosphate131

e)D-Glucose-6-phosphate and D-fructose-6-phosphate134

f)D-Gluconic acid139

g)D-6-Phosphogluconate143

h)D-Glucosamine146

i)D-Glucosamine-6-phosphate151

k)D-Fructose156

l)D-Fructose-1,6-diphosphate160

Determination with FDPase160

m)L-Sorbose-6-phosphate164

n)D-Sorbitol167

o)myo-Inositol171

p)D-Ribose-5-phosphate175

q)L-Ribulose and L-arabinose178

r)D-Ribulose182

s)D-Ribulose-5-phosphate186

t)D-Ribulose-1,5-diphosphate188

u)L-Xylulose191

v)D-Xylulose194

Determination with DPN-xylitol dehydrogenase194

w)D-Xylulose and D-xylose196

Determination with D-xylose isomerase196

x)D-Xylulose-5-phosphate201

y)D-Erythrose-4-phosphate205

z)L-Erythrulose208

3．Three-Carbon Compounds211

a)Glycerol211

b)L-Glycerol-1-phosphate215

c)D-Glycerate220

d)D-3-Phosphoglycerate,D-2-phosphoglycerate,phosphoenolpyruvate224

e)D-1,3-Diphosphoglycerate234

f)D-2,3-Diphosphoglycerate238

g)L-Glyceraldehyde-3-phosphate241

h)Dihydroxyacetone244

i)Dihydroxyacetone phosphate,D-fructose-1,6-diphosphate and D-glyceraldehyde-3-phosphate246

k)Pyruvate253

l)Hydroxypyruvate260

m)β-Mercaptopyruvate263

n)L-Lactate266

Determination with LDH and DPN266

Determination with yeast LDH271

Determination with LDH and AP-DPN275

o)D-Lactate278

p)Methylglyoxal283

4．Two-and One-Carbon Compounds285

a)Ethanol285

Determination with ADH and DPN285

Determination with ADH and AP-DPN287

b)Acetaldehyde290

Determination with ADH290

Determination with aldehyde dehydrogenase292

c)Glycolaldehyde297

d)Glyoxylic acid300

e)Acetate303

f)Formate308

Ⅱ．Substances Involved in the Citric Acid Cycle313

a)Citrate313

Determination with citrase313

b)Citrate and isocitrate318

Determination with aconitase and isocitric dehydrogenase318

c)α-Oxoglutarate324

d)L-Malate328

Determination with MDH and DPN328

Determination with MDH and AP-DPN332

e)Oxaloacetate335

f)Succinate340

g)Fumarate344

Ⅲ．Substances Involved in Protein Metabolism350

1．Proteins and Peptides350

a)Characterization of peptides and proteins with enzymes350

b)Glutathione363

2．Amino Acids,Amines and Amides367

a)D-Amino acids367

b)L-Amino acids(Lys,Arg,Orn,Tyr,His,Glu,Asp)373

c)L-Alanine378

d)L-Aspartic acid and L-asparagine381

e)L-Glutamic acid(and L-glutamine)384

Determination with GIDH384

Determination with GIDH and LDH388

Determination with GIDH and AP-DPN392

Determination with GPT394

f)DL-Threonine398

g)Urea401

h)Creatine407

Ⅳ．Substances Involved in the Metabolism of Fatty Acids,Lipids and Steroids411

a)Polyunsaturated fatty acids411

b)Lecithin415

c)Acyl-CoA derivatives419

Acetyl-CoA419

Acetoacetyl-CoA425

Acrylyl-CoA429

Benzoyl-CoA431

Butyryl-CoA and CoA derivatives of the higher saturated fatty acids433

Crotonyl-CoA437

L-(+)-β-Hydroxybutyryl-CoA441

β-Hydroxy-β-methylglutaryl-CoA445

β-Hydroxypropionyl-CoA449

Malonylsemialdehyde-CoA452

d)Acetoacetate454

e)D-(－)-β-Hydroxybutyrate459

f)Hydrolysis of steroid conjugates462

g)20-Ketosteroids477

Spectrophotometric method477

Fluorimetric method480

h)Steroid alcohols in urine485

Ⅴ．Nucleosides,Purines and Pyrimidines,Coenzymes and Related Substances491

1．Nucleosides,Purines and Pyrimidines491

a)Adenosine491

b)Hypoxanthine and xanthine495

c)Uric acid500

d)Inosine502

e)Guanosine505

f)Orotic acid508

2．Coenzymes and Related Substances512

a)Coenzyme A512

Determination of CoA-SH and CoA-S-S-CoA with HOADH513

Determination of CoA-SH with PTA517

Determination of CoA-SH with TK520

Determination of CoA-SH with CCE523

b)Diphosphopyridine nucleotide528

c)Reduced diphosphopyridine nucleotide531

d)Triphosphopyridine nucleotide535

e)Reduced triphosphopyridine nucleotide537

f)Adenosine-5′-triphosphate539

Determination with PGK539

Determination with HK/G6P-DH543

Determination by enzymatic fluorimetry551

g)ATP and creatine phosphate559

Determination with luciferase559

h)Adenosine-5′-diphosphate and adenosine-5′-monophosphate573

i)Adenosine phosphates578

k)UDP-glucose,UDP-galactose,UTP,UDP-glucuronic acid581

l)Flavine adenine dinucleotide596

m)Flavine mononucleotide599

n)Thiamine pyrophosphate602

o)Pyridoxal phosphate606

p)Creatine phosphate610

Ⅵ．Other Substrates and Miscellaneous Substances617

a)Organophosphorus insecticides617

b)DDT626

c)Inorganic peroxides633

d)Nitrate636

e)Magnesium in plasma640

f)Pyrophosphate644

Section C:Measurement of Enzyme Activity Preliminary Remarks651

Ⅰ．The Importance of the Measurement of Enzyme Activity651

a)In medicine651

b)In food chemistry713

c)In botanical and agricultural chemistry720

Ⅱ．Methods724

1．Aldolases724

a)Fructose-1,6-diphosphate aldolase724

Colorimetric determination with 2,4-dinitrophenylhydrazine725

Determination with enzymatic auxiliary and indicator reactions728

b)1-Phosphofructoaldolase732

2．Dehydrogenases736

a)Lactic dehydrogenase736

b)Glucose-6-phosphate dehydrogenase744

c)Glutamic dehydrogenase752

d)Malic dehydrogenase757

e)Sorbitol dehydrogenase761

3．Esterases765

a)Acetylcholinesterase765

b)Cholinesterases771

Colorimetric determination772

Manometric determination774

c)Lipase776

d)Phosphatases(phosphomonoesterases)779

Determination in serum with phenolphthalein diphosphate779

Determination in serum with p-nitrophenylphosphate783

Determination in milk with phenylphosphate785

e)Glucose-6-phosphatase788

f)Ribonuclease793

Spectrophotometric method794

Titrimetric method797

4．Proteases800

a)Chymotrypsin800

Determination with casein as substrate800

Determination with L-tyrosine ethyl ester as substrate802

b)Trypsin807

Determination with haemoglobin as substrate808

Determination with casein as substrate811

Determination with benzoylarginine ethyl ester as substrate815

c)Pepsin819

d)Peptidases824

Acidimetric determination of dipeptidases824

Colorimetric determination of carboxypeptidase828

Colorimetric determination of leucine aminopeptidase in serum830

5．Transaminases837

a)Glutamate-oxaloacetate transaminase837

Determination with MDH as indicator enzyme837

Colorimetric determination with 2,4-dinitrophenylhydrazine842

b)Glutamate-pyruvate transaminase846

Determination with LDH as indicator enzyme846

Colorimetric determination with 2,4-dinitrophenylhydrazine851

6．Others854

a)Amylase854

b)Creatine phosphokinase859

c)Galactose-1-phosphate uridyl transferase863

d)β-Glucosidase867

e)β-Glucuronidase869

f)Glutathione reductase875

TPNH-specific glutathione reductase876

DPNH-specific glutathione reductase877

g)Kallikrein880

Determination of kallikreins880

Determination of kallikrein inhibitors883

h)Catalase885

Spectrophotometric method886

Determination by iodometric titration888

Determination with the Katalaser891

i)Peroxidase895

k)Reductases898

l)Inveftase901

Determination in food chemistry901

Determination in botanical and agricultural chemistry904

m)Thromboplastin time908

Determination according to Quick909

＂Geigy＂micromethod910

n)Urease913

Titrimetric method913

Colorimetric method915

o)Xanthine oxidase917

Ⅲ．Histochemical Detection of Enzymes920

1．Introduction(Aims of the Method)921

2．General923

a)Principle techniques923

b)Requirements924

c)Sources of error925

d)Range of application925

e)Precautions necessary for the preservation of cell components925

3．Special Methods929

a)Methods involving precipitation of metallic salts929

b)Methods employing azonaphthol dyes943

c)Methods employing indigo dyes949

d)Methods employing indoaniline and azamethine dyes950

e)Methods employing tetrazolium salts953

f)Detection of polysaccharide synthesis609

Section D:Biochemical Reagents967

Ⅰ．Enzymes968

Ⅱ．Coenzymes and Substrates1002

Ⅲ．Complete Reagent Kits1034

Tables1039

Conversion Table:Transmittance？Extinction1041

Atomic Weights1045

Index1047