Biochemical Reagents

Biochemical Reagents

Hans-Ulrich Bergmeyer, Helmut Klotzsch, Hans Mollering, Michael Nelbock-Hochstetter and Klaus Beaucamp

Enzymes, coenzymes and metabolites are the reagents characteristic of enzymatic analysis.

Some of these materials must be prepared or isolated in the laboratory, but many are avail­

able commercially. Information about these materials is less readily available to the analyst than information about inorganic reagents. This situation is made worse by the multiplicity and variety of the definitions of purity. In 1955, the Committee on Biological Chemistry and Chemical Technology, National Academy of Sciences, National Research Council (USA) began to collect standard values for biochemical compounds 1 ). As a result of this work the first volume of "Specifications and Criteria of Biochemical Compounds" was published in June I960*). This volume does not contain chapters on "Phosphate Esters" and "Enzymes".

Biochemical reagents are described below if they are important for enzymatic analysis. The

"Specifications and Criteria" is referred to by the abbreviation NAS-NRC (National Aca­

demy of Sciences — National Research Council).

Sources of commercial preparations have been taken from the catalogues in existence in October 1961 and the compilation is not claimed to be exhaustive.

On the whole manufacturers rather than retailers are named; addresses are listed in Table 1.

Table 1. The main manufacturers and retailers of biochemical reagents.

Boehringer

British Drug Houses California Corporation

Cutolo Calosi

General Biochemicals Inc.

Light

Mycofarm-Delft

Nutritional Biochemicals Corp.

Pabst Schwarz Seravac Sigma Worthington

C. F. Boehringer & Soehne G m b H , Mannheim (Germany) British Drug Houses Ltd., Poole (England)

California Corporation for Biochemical Research, Los Angeles, Calif. ( U S A )

Farmochimica Cutolo Calosi, Naples (Italy)

General Biochemicals Inc., Chagrin Falls, Ohio ( U S A ) Koch-Light Laboratories Ltd., Colnbrook, Bucks, (England) Mycofarm-Delft, Division of Royal Netherlands Fermen­

tation Industries Ltd., Delft (Holland)

Nutritional Biochemicals Corp., Cleveland 28, Ohio ( U S A ) Pabst Laboratories, Division of Pabst Brewing Company, Milwaukee 5, Wise. ( U S A )

Schwarz Bioresearch, Inc., Orangeburg, N . Y. ( U S A ) Seravac Laboratories PTY. Ltd., Cape T o w n (South Africa) Sigma Chemical Company, St. Louis 18, M o . ( U S A ) Worthington Biochemical Corporation, Freehold, N.J. ( U S A )

*) Publication 719 of the National Academy of Sciences — National Research Council; available from: Printing and Publishing Office, National Academy of Sciences — National Research Council. 2101 Constitution Ave., N . W., Washington 25, D . C , U S A .

•)//.£. Carter, J. T. Edsall, S. Weinhouse, H. Neurath, O. Schales and C. V. Smythe, Science 123, 54 [1956].

I. Enzymes

For the characterization and standardization of an enzyme, information is required about its source, the reaction it catalyses, kinetic data, the most important activators and inhibitors, as well as basic physical and physico-chemical values.

The best criterion for the purity of an enzyme is the specific activity together with details of the activity of contaminating enzymes. The activity of these contaminants is expressed as a percentage of the specific activity of the enzyme being described.

The activities of the enzymes are given in units and the specific activity in units per mg.

protein. To conform to the proposed International Unit (see p. 32), a unit of enzyme acti­

vity is defined as the amount of enzyme which transforms 1 (jimole of substrate in 1 min.

at 25° C. When other units are given they are defined.

Only the most important references for the isolation and purification as well as for the deter­

mination of the activity of the enzyme are given below. The choice of reference has been governed by the experience of the authors. Only the important physical and chemical data on the enzymes and on their activators and inhibitors are given. In addition to the trivial names of the enzymes the official designation according to 2) is given with the reference

"IUB nomenclature".

Adenosine Deaminase (ADA) from Calf Intestine Adenosine aminohydrolase *>

Adenosine + H2O > inosine -f N H 3

A D A has not yet been crystallized. For the isolation of a highly purified enzyme, see e.g.^.

The molecular weight is not known. p H Optimum: 6.5 to 7.7 (dependent on the salt concentration).

The reaction catalysed by the enzyme is irreversible. The Michaelis constants are not known.

Activators, Inhibitors, Chemical Properties:

Inosine does not inhibit the reaction. 8 M Urea has an inhibitory action; this inhibition is dependent on temperature and p H . 4 x 1 0

-4

-7

bited by p-chloromercuribenzoate.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

4

Stability:

Suspensions in 2.7 M ( N H 4 )

2

Enzyme solutions or suspensions can be dialysed against 0.01 M phosphate buffer (pH 6.0) at 0 to 4°C for 12 hours.

*> I U B nomenclature

2) Report of the Comission on Enzymes of the International U n i o n of Biochemistry 1961. I.U.B.

Symposium Series Vol. 20. Pergamon Press, L o n d o n 1961.

3) W. Klein, Hoppe-Seylers Z. physiol. Chem. 324, 163 [1961].

4

Purity Required:

A universally suitable ADA preparation should contain ca. 150 units/mg. Contaminants: not more than 0.01 % alkaline phosphatase, myokinase, nucleoside phosphorylase, 5'-AMP deaminase, guanase and xanthine oxidase.

Commercial Preparations:

Boehringer (suspension as described under "Stability"). — Sigma (dry powder).

Alcohol Dehydrogenase (ADH) from Yeast Aldehyde reductase; alcohol: NAD oxidoreductase *) C 2 H 5 OH + DPN+ , 1 CH3CHO + DPNH + H+

ADH crystallizes in fine needles from

M (NH^SC^ solution. For the isolation, see

5

Molecular weight:

Michaelis constants:

M f or DPN;

for ethanol;

2.3 X 10-5 M for DPNH; 1.1 x 10-4 M for acetaldehyde at pH 7.9 and 26°C.

A c t i v a t o r s , Inhibitors, C h e m i c a l Properties:

ADH contains up to 40 SH groups and 5 zinc atoms per molecule; it is therefore sensitive to oxidizing and chelating agents.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity 6 ) at 340 or 366 mu. with ethanol and DPN (see the information leaflets of various firms, e.g. Boehringer and Worthington).

Stability:

The crystalline suspension in 2.0 M (NH 4 ) 2 S04 solution containing 3 % Na4P 2 0 7 and 1 % glycine (ca. pH 8) is stable for several months at 0 to 4°C when air is excluded, while at room temperature it is only stable for 1 week. Solutions or suspensions can be dialysed against distilled water at 0 to 3°C without any significant loss of activity.

Purity Required:

A universally suitable ADH preparation should contain ca. 180 units/mg. Contaminants: not more than 0.05% carboxylase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase;

0.03% myokinase; 0.02% aldolase; 0.01 % pyruvic kinase and lactic dehydrogenase.

Commercial Preparations:

Boehringer (crystalline suspension in (NH4) 2 SC>4 solution as described under "Stability"). — Myco- farm-Delft (crystalline suspension in 2 M (NH4) 2 SC>4 solution containing 2 % Na4P 2 C>7 and 0.5%

glycine). Sigma (dry powder and crystalline suspension). — Worthington (dry powder containing 75% protein; crystalline suspension in 60% saturated (NH4) 2 SC>4 solution containing 3 % Na4P 2 C>7 and 0.1 % glycine).

*) I U B nomenclature

5) E. Racker, J. biol. Chemistry 184, 313 [1950].

6) analogous to Th. Biicher and H. Redetzki, Klin. Wschr. 29, 615 [1951].

Aldolase (ALD) from Skeletal Muscle (Rabbit) Ketose-1 -phosphate aldehyde-lyase *)

D-(+)-Fructose-l,6-diphosphate ^ * dihydroxyacetone phosphate +

D-(+)-glyceraldehyde-3-phosphate According to the anion and the p H value A L D crystallizes from salt solutions in different forms. It crystallizes as hexagonal bipyramids from 1.6 M a m m o n i u m sulphate solution (ca. p H 6), while at p H 7.5 it crystallizes as needles. For the isolation, see e.g.

1

Molecular weight: ca. 148000; isoelectric point: 6.1 (phosphate buffer, ionic strength 0 . 1 ; dependent on the buffer and the ionic strength).

Activators, Inhibitors, Chemical Properties:

Heavy metal ions inhibit, for example, 2 x 1 0 ~

4

2+

+

Blocking the first 10 SH groups has no affect on the activity. A 30 to 7 0 % inhibition occurs when 12 or 14 SH groups are blocked. Glutathione reactivates A L D when not more than 25 SH groups are blocked. Iodine inhibits, but iodoacetate does not.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay

7

cator enzyme (see the information leaflets of various firms, e.g. Boehringer).

Stability:

Crystalline suspensions (hexagonal pyramids) are stable in 2.0 M ( N H ^ S C ^ solution (ca. p H 6) for years at 0 to 4°C. Before use carefully suspend the crystals. Solutions or suspensions can be dialysed against 200 volumes 10

3

Purity Required:

A universally suitable A L D preparation should contain ca. 9 units/mg. Contaminants: not more than 0.05% lactic dehydrogenase and glycerol-1-phosphate dehydrogenase; 0 . 0 1 % pyruvic kinase and glyceraldehyde-3-phosphate dehydrogenase; 0 . 0 3 % triosephosphate isomerase.

Commercial Preparations:

Boehringer (crystalline suspension as described under "Stability")- — Sigma (crystalline suspension).

Carboxypeptidase A**) from Pancreas

The enzyme hydrolyses terminal carboxyl amino acids (except proline) from peptides.

Carboxypeptidase crystallizes as plates from salt-free, aqueous solution. For the isolation of the cry­

stalline enzyme, see e.g. 8).

Molecular weight: 3 4 3 0 0 ; p H optimum ca. 7.5; isoelectric point: p H 6.0; Michaelis constant (25°C) K

M

-2

-3

*) I U B nomenclature

**) N o designation has so far been proposed by the IUB.

7) G. Beisenherz, H. J. Boltze, Th. Bucher, R. Czok, K. H. Garbade, E. Meyer-Arendt and G. Pflel- derer, Z. Naturforsch. 8b, 555 [1953].

8) M. L. Anson, J. gen. Physiol. 20, 663 [1937].

Activators, Inhibitors, Chemical Properties:

Cobalt ions activate the enzyme. Carboxypeptidase A contains 1 mole of zinc per mole enzyme.

The Zn can be removed by dialysis against o-phenanthroline solution, resulting in loss o f activity. T h e metal-free apoenzyme can be reactivated by Zn, M n , Fe, C o and N i . Citrate, oxalate, pyrophosphate, cysteine, iodoacetate and indoleacetate are inhibitors of the enzyme. 1 0 ~

4

D e t e r m i n a t i o n of A c t i v i t y

9 )

The activity is determined by hydrolysis o f carbobenzoxyglycyl-L-phenylalanine and colorimetric estimation o f the amino acid liberated with ninhydrin

1 0

Stability:

Aqueous suspensions stabilized with toluene keep for several months at 0 to 4 ° C . Solutions in 1 0 % LiCl solution are stable for several weeks. Solutions in bicarbonate, phosphate and tris buffer are unstable.

Purity Required:

A universally suitable preparation should have a proteolytic coefficient Q of at least 10 (see p. 357).

Trypsin and chymotrypsin, which may occur as contaminants, are inhibited by diisopropylfluoro­

phosphate.

Commercial Preparations:

Worthington, Sigma, California Corporation (suspension in water, stabilized with toluene).

Catalase (CAT) from Beef Liver

2 H

2

2

2

2

Catalase crystallizes as needles from aqueous solutions at p H 6. For the isolation, see e.g. n ) . Molecular weight: 248000. Under the conditions of the assay of activity the cleavage of peroxides by catalase is a first order reaction. Range of activity: p H 4 to 9 with the optimum at p H 6.8. T h e opti­

m u m temperature is 4 0 ° C (previously assumed to be: 0 to 10°C).

Activators, Inhibitors, C h e m i c a l Properties:

N o activators are known. C u

2 +

2

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity at 240 mu. with H

2

2

1 2

*) I U B nomenclature

9) H. Neurath, E. Elkins and S. Kaufman, J. biol. Chemistry 170, 221 [1947].

10) S. Moore and W. H. Stein, J. biol. Chemistry 176, 367 [1948].

n

Academic Press, N e w York 1955, Vol. II, p. 775.

12) H.-U. Bergmeyer, Biochem. Z. 327, 255 [1955].

Stability:

Crystalline suspensions in water containing 0.5% thymol or solutions in 3 0 % glycerol are stable for longer than a year at 0 to 4 ° C . Solutions can be incubated at 30° C for several days without signi­

ficant loss of activity. On freezing and thawing, catalase solutions lose relatively large amounts of activity.

Purity Required:

A universally suitable catalase preparation should contain ca. 39000 units/mg. *). The preparation must be free from interfering enzyme contaminants. For some purposes purified preparations are suitable.

Commercial Preparations:

Boehringer (suspension or solutions as described under " Stability"). Sigma; Paul Lewis Laboratories, Inc., Milwaukee, U S A ; The Armour Laboratories, Kankakee, 111., U S A ; Worthington (in various grades as solutions, suspensions and dry powder).

a-Chymotrypsin (CHTR)**) from Pancreas

The enzyme hydrolyses peptides, amides and esters. Bonds containing the carboxyl group of aromatic L-amino acids are particularly easily hydrolysed.

a-Chymotrypsin crystallizes as fine needles or rosettes from 2 0 % alcoholic solution (pH 4.0). For the isolation of the crystalline enzymes after activation of a-chymotrypsinogen with trypsin, see e.g. '3) Molecular weight: ca. 2 5 0 0 0 ; p H optimum: 7.0 to 9.0; isoelectric point: ca. 8.3.

A c t i v a t o r s , Inhibitors, Chemical Properties:

Calcium ions activate the enzyme. Diisopropylfluorophosphate reacts with the serine hydroxyl groups of the enzyme; the inactive product can be crystallized like a-chymotrypsin. a-Chymotrypsin is reversibly denatured by heat, urea and extremes of p H . Heavy metals cause an irreversible inhibition.

D e t e r m i n a t i o n of A c t i v i t y

1 4

Spectrophotometric assay of activity at 237 mu. by the measurement of hydrolysis of A-acetyl-L- tyrosine ethyl ester (see the information leaflets of various firms, e.g. Seravac, Worthington) or with casein as substrate according to Kunitz

15

Stability:

Solutions at p H 3.0 are stable. At higher p H the activity slowly decreases even in the cold. Dry preparations are stable for years if stored, stoppered, in the cold. Solutions can be dialysed against 10-3 M H C 1 a t O t o 4 ° C .

Purity Required:

A universally suitable preparation should contain 8 0 0 0 to 10000 units ***)/mg. according to

1 4

corresponding to ca. 6 units/mg. according t o

1 5

1 6

*) This corresponds to ca. 3 0 0 0 units/mg. according t o

1 2

**) So far no designation has been proposed by the I U B .

***) A unit is the amount of enzyme in 3 ml., which causes a decrease of optical density at 237 mu.

of 0.001/min. with 7V-acetyl-L-tyrosine ethyl ester as substrate and at p H 7.0 and 25° C.

13) M. Kunitz and / . H. Northrop, J. gen. Physiol. 79, 991 [1936].

1

4

15) M. Kunitz, J. gen. Physiol. 30, 291 [1947].

16) F. C. Wu and M. Laskowski, J. biol. Chemistry 213, 609 [1955].

Commercial Preparations:

Boehringer, California Corporation, Seravac, Sigma, Worthington (various grades, dry powder).

Creatine Phosphokinase (CPK) from Skeletal Muscle (Rabbit)

Creatine kinase; ATP-creatine-transphosphorylase; ATP: creatine phosphotransferase *)

Creatine + A T P ^

N

C P K crystallizes as fine needles from 6 0 % ethanol, p H 9.0

(NH4OH).

1 7 )

2

Activators, Inhibitors, C h e m i c a l Properties:

M g

2+

2+

2+

2 +

2 +

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

1 8

Stability:

If stored as a powder in the dry state C P K is stable for over 1 year at 0 to 4° C. C P K solutions can be dialysed against 0.05 M a m m o n i u m citrate solution, p H 9.0 at 0 ° C with little loss of activity.

Purity Required:

A universally suitable preparation should contain ca. 10 units/mg. Contaminants: not more than 1 % triose phosphate isomerase; 0 . 0 3 % phosphoglucose isomerase and glutathione reductase; 0 . 0 1 % lactic dehydrogenase, pyruvic kinase, myokinase and ATPase.

Commercial Preparations:

Boehringer (salt-free, lyophilized powder, obtained from crystalline CPK). — Sigma (like the preced­

ing preparation).

Enolase from Skeletal Muscle (Rabbit)

Phosphopyruvate hydratase; D-2-phosphoglycerate hydro-lyase *>

D-2-Phosphoglycerate

v N

Enolase crystallizes as very fine needles from 2.4 M a m m o n i u m sulphate solution (pH 5.0). For the isolation, see e.g.

l 9

Molecular weight: not k n o w n ; Michaelis constants: 5.8 x l O

-5

9 . 2 x l O

_ 5

4

2 +

6.2 to 6.9.

*) I U B nomenclature

17) S. A. Kuby, L. Noda and H. A. Lardy, J. biol. Chemistry 209, 191 [1954].

is) M. L. Tanzer and C. Gdvarg, J. biol. Chemistry 234, 3201 [1959].

19) R. Czok and Th. BUcher, Advances in Protein Chemistry 15, 373 [I960].

Activators, Inhibitors, Chemical Properties:

Muscle enolase is extremely specific for M g

2 +

2 +

2

2+

4

- 4

1 0

-3

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay at 240 mu. with phosphoenolpyruvate or at 340 or 366 mu. with D-2-phospho- glycerate (see p. 229). Pyruvic kinase is used as the auxiliary enzyme and lactic dehydrogenase as the indicator enzyme (see the information leaflets of various firms, e.g. Boehringer).

Stability:

Crystalline suspensions in 2.6 M ( N H

4

2

-3

Purity Required:

A universally suitable preparation should contain ca. 25 units/mg. Contaminants: not more than 0.1 % pyruvic kinase; 0 . 0 2 % phosphoglycerate mutase and lactic dehydrogenase; 0.01 % glyceralde- hyde-3-phosphate dehydrogenase and phosphoglycerate kinase.

Commercial Preparations:

Boehringer (crystalline suspension as described under "Stability"). — Sigma (suspension).

Glucose Oxidase (GOD) from Penicillium notatum P-D-Glucose: O2 oxidoreductase *>

P-D-Glucose + H2O + 0

2

G O D crystallizes as needles from 2.6 M a m m o n i u m sulphate solution (ca. p H 5.0). For the isolation of the crystalline enzyme from Penicillium amagasakiense, see e.g.

20

Molecular weight: ca. 1 5 0 0 0 0 ; p H o p t i m u m : 5 . 6 ; Absorption m a x i m a : at 2 7 5 , 3 3 7 and 4 5 1 mu.;

Michaelis constant for glucose: 9.6 x 1 0 ~

3

2

A c t i v a t o r s , Inhibitors, Chemical Properties:

2,6-Dichlorophenolindophenol, thionine, quinone and methylene blue can replace oxygen as the hydrogen acceptor. The enzyme is not inhibited by CO, H C N and sodium azide nor by N a F or urethane. 10~

2

10~

3

3

G O D contains 2 moles F A D per mole.

D e t e r m i n a t i o n of A c t i v i t y :

Manometric assay of activity 20) at 30° C and p H 5.6 with glucose and oxygen, and addition of catalase (see the information leaflets of various firms, e.g. Boehringer).

Stability:

Dry preparations are stable virtually indefinitely. Solutions between p H 3.5 and 8.0 are stable for several weeks at 0 t o 4° C . Enzyme solutions can be dialysed against distilled water at 0 t o 4° C without significant loss of activity.

*) IUB nomenclature

2

Purity Required:

For the methods described in this book a G O D preparation containing ca. 14 units/mg. is suitable;

this corresponds to a Q o

2

Commercial Preparations:

Boehringer (lyophilized powder). — Sigma (lyophilized powder).

Glucose-6-phosphate Dehydrogenase (G6P-DH)

G 6 P - D H crystallizes as bipyramids from a solution of 2.5 M ( N H

4

2

4

Non-crystalline, but highly purified preparations are available commercially or can be prepared according t o

2 2

2 3

Molecular weight: has not yet been determined; Michaelis constants at p H 8.0 and 25°C in 0.01 M M g C l

2

2

5

Activators, Inhibitors, Chemical Properties:

Activation by M g

2+

T P N H and glucosamine-6-phosphate inhibit. G 6 P - D H is extremely sensitive to heavy metal ions.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric a s s a y

2 2

Stability:

Suspensions in 3.3 M ( N H

4

2

4

4

Purity Required:

A universally suitable preparation need not be crystalline, but should contain at least 70 units/mg.

Contaminants: not more than 0.5% glutathione reductase; 0.4% hexokinase; 0 . 1 % 6-phospho­

gluconic dehydrogenase; 0.05% phosphoglucose isomerase.

Commercial Preparations:

Boehringer (suspension in 3.3 M ( N H

4

2

4

4

2

4

^-Glucuronidase and Aryl Sulphatase

(Helix pomatia)

P- D - G l u c u r o n i d e g l u c u r o n o h y d r o l a s e *) a n d s t e r o l - s u l p h a t e s u l p h o h y d r o l a s e *)

^-glucuronidase

R-Glucuronide + H

2

R-Sulphate + H

2

Aryl sulphatase hydrolyses sulphate esters from phenols; ^-glucuronidase hydrolyses aliphatic and aromatic ^-glucuronides. So far neither enzyme has been crystallized. ^-Glucuronidase has been purified from liver, kidney, brain, bacteria and molluscs. For the isolation of the two enzymes from snails, see e.g.

24

*) I U B nomenclature

20 E. A. Noltmann, C. J. Gubler and S. A. Kuby, J. biol. Chemistry 236, 1225 [19611.

22) L. Glaser and D. H. Brown, J. biol. Chemistry 216, 67 [1955].

23) / . Cooper, P. A. Srere, M. Tabachniek and E. Racker, Arch. Biochem. Biophysics 74, 306 [1958].

p H Optimum of (3-glucuronidase (Helix pomatia): 4.4 to 4.8 with phenolphthalein-p-glucuronide as substrate. Michaelis constants: 5 . 7 x l O ~

5

4

Activators, Inhibitors, Chemical Properties:

The activity of p-glucuronidase is increased by deoxyribonucleic acid and numerous diamines.

2.5 x 1 0

-6

D e t e r m i n a t i o n of A c t i v i t y :

^-Glucuronidase with phenolphthalein (3-glucuronide as substrate at p H 4.5 and 3 8 ° C . The phenol­

phthalein liberated in 1 hour is measured colorimetrically at 546 m o .

2 5

Aryl sulphatase with phenolphthalein disulphate as substrate at p H 6.2 and 38° C. The phenolphthalein liberated in 1 hour is measured colorimetrically at 546 m u .

2 6

Stability:

Enzyme solutions (ca. p H 6.0) stabilized with butanol scarcely lose any activity in a year if stored, stoppered, at 0 to 4 ° C .

Purity Required:

A universally suitable preparation should contain ca. 100000 Fishman units/ml. (1 Fishman unit is the amount of enzyme which liberates 1 pig. phenolphthalein from phenolphthalein (3-glucuronide in 1 hour at p H 4.5).

Aryl sulphatase should contain ca. 50000 Whitehead units/ml. (1 Whitehead unit is the amount of enzyme which liberates 1 u.g. phenolphthalein from phenolphthalein disulphate in 1 hour at p H 6.2).

Commercial Preparations:

Boehringer (enzyme solution from Helix pomatia as described under "Stability"). — Industrie Bio- logique Francaise S. A., Paris (enzyme solution from Helix pomatia as described under " Stability "). — Sigma (^-glucuronidase: dry powder from bacteria, liver, Patella; sulphatase: dry powder from Patella). — Seravac (lyophilized p-glucuronidase from molluscs, patella barbara Linnaeus). — Worthington (salt-free, lyophilized p-glucuronidase from liver).

Glutamate-Oxaloacetate Transaminase (GOT) from Pig Heart L-Aspartate : 2-oxoglutarate aminotransferase *>

L-Glutamate + oxaloacetate ^ ^ a-oxoglutarate + L-aspartate G O T has not yet been crystallized. For the isolation, see e.g.

21

Molecular weight: ca. 110000; p H o p t i m u m : CA. 8.3; Michaelis constants: have not yet been measured.

Activators, Inhibitors, Chemical Properties:

0.05 M C N " causes a 8 0 % inhibition, while with 0.001 M C N ~ it is 30%. At a concentration of 3.5 x l O

-2

*) I U B nomenclature

24) K. S. Dodgson and B. Spencer, Biochem. J. 55, 315 [1953].

25) p. Talalay, W. H. Fishman and C. Huggins, J. biol. Chemistry 166, 757 [1946].

26) E. M. Whitehead, A. R. Morrison and L. Young, Biochem. J. 57, 585 [1952].

27) W. T. Jenkins, D. A. Yphantis and / . W. Sizer, J. biol. Chemistry 234, 50 [1959].

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity at 340 or 366 mpi with L-aspartate and a-oxoglutarate (see p. 837).

Malic dehydrogenase is used as the indicator enzyme (see the information leaflets o f various firms, e.g. Boehringer).

Stability:

Suspensions in 3 M ( N H

4

2

-3

2

Purity Required:

A universally suitable G O T preparation should contain at least 180 units/mg. Contaminants: not more than 0.05% glutamate-pyruvate transaminase and oxaloacetate decarboxylase; 0 . 0 1 % lactic dehydrogenase, malic dehydrogenase and glutamic dehydrogenase.

Commercial Preparation:

Boehringer (suspension as described under "Stability").

Glutamate-Pyruvate Transaminase (GPT) from Pig Heart L-Alanine : 2-oxoglutarate aminotransferase *)

L-Alanine + a-oxoglutarate ^ * pyruvate -h L-glutamate G O T has not yet been crystallized. For the isolation, see e.g.

28

Molecular weight: 180000; p H o p t i m u m : in 0.1 M phosphate buffer ca. 8.2; in 0.1 M tris buffer ca. 7.6.

Activators, Inhibitors, Chemical Properties:

G P T contains 5 S H groups and a maximum of 0.9 moles pyridoxal phosphate per 100000 g. protein and is therefore yellow in weakly acid solution. /?-Chloromercuribenzoate inhibits the enzyme com­

pletely; this inhibition can be partially reversed with cysteine and pyridoxal phosphate. 10~~

2

-3

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity at 340 or 366 mu, with L-alanine and a-oxoglutarate (see p. 846);

lactic dehydrogenase is used as the indicator enzyme (see the information leaflets o f various firms, e.g. Boehringer).

Stability:

Suspensions in a solution of 1.7 M ( N H

4

2

4

_ 2

_ 3

glutarate are stable for many months at 0 to 4 ° C . Solutions or suspensions can be dialysed against 200 volumes 0.01 M potassium phosphate buffer (pH 7.6) for 4 hours at 0 to 3°C.

Purity Required:

A universally suitable G P T preparation should contain ca. 25 units/mg. Contaminants: not more than 0.05% glutamate-oxaloacetate transaminase; 0 . 0 2 % malic dehydrogenase; 0 . 0 1 % lactic dehydrogenase. Glutamic dehydrogenase should not be detectable.

*) I U B nomenclature

28) L. Grein and G. Pfleiderer, Biochem. Z. 330, 433 [1958].

Commercial Preparation:

Boehringer (suspension as described under "Stability").

L-Glutamic Dehydrogenase (GIDH) from Beef Liver L-Glutamate: NAD oxidoreductase (deaminating) *>

L-Glutamate + H

2

4+

G I D H crystallizes as fine needles (ca. 5 u. long) from ca. 1.0 M a m m o n i u m sulphate solution (pH 7.4).

For the isolation of the crystalline enzyme, see e.g.

29

Molecular weight: 1 0 0 0 0 0 0 ; p H o p t i m u m : 7.6 to 8.5 (dependent on the buffer); equilibrium constant [ G l u ] [ D P N + ] [ H

2

K

4

3

8 X I 0 1 4 1

M o 1

Michaelis constants: l . O x 1 0 ~

4

3

7.0 X 10-4 M for a-oxoglutarate; 5.6 x 1 0

-2

4

Cysteine protects the enzyme from inactivation by heat. In low concentration thioglycollic acid, p-mercaptoethylamine, E D T A and a.a'-dipyridyl have an activating effect. The reaction is competi­

tively inhibited by D-glutamate, glutamine, aspartate and hydroxylamine. 1 X 1 0

-4

2

Other inhibitors are: N a N 3 thyroxine, heparin, sulphonylurea and all heavy metals (e.g. 1 0

-5

2+

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric determination of activity

3

Stability:

Suspensions in 2.0 M ( N H

4

2

4

Purity Required:

A universally suitable G I D H preparation should contain ca. 3 units/mg. (oxidative deamination of L-glutamate). A m m o n i u m ions interfere in s o m e reactions; in this case use a solution o f the dialysed enzyme in 5 0 % glycerol. Contaminants: not more than 0 . 1 % catalase, glycerol-1-phosphate dehydrogenase and alcohol dehydrogenase.

Commercial Preparations:

Boehringer (suspension and solution as described under "Stability"). — Sigma (suspension in am­

monium sulphate solution).

*) I U B nomenclature

29) / . A. Olson and C. B. Anfinsen, J. biol. Chemistry 797, 67 [1952].

30) H. J. Strecker, Arch. Biochem. Biophysics 46, 128 [19531.

Glutathione Reductase (GR) from Yeast NAD(P)H2 : glutathione oxidoreductase *)

G S S G (oxidized glutathione) + T P N H + H+ > 2 G S H (glutathione) + T P N + Pure glutathione reductase from yeast has not yet been crystallized. For the isolation of a purified preparation, see e.g.*

1

Molecular weight: the molecular weight of the purified yeast enzyme has not yet been determined.

Activators, Inhibitors, Chemical Properties:

The enzymatic activity is not dependent on the presence of individual ions. Heavy metal ions, for example, Z n

2 +

2 +

2 +

Spectrophotometric assay of activity

3 1

Stability:

Suspensions in 2.8 M ( N H

4

2

NH4OH

Purity Required:

A universally suitable G R preparation should contain ca. 90 units/mg. Contaminants: not more than 1 % D P N H - d e p e n d e n t G R ; 0.1 % glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydro­

genase, isocitric dehydrogenase and T P N H oxidase.

Commercial Preparations:

Boehringer (suspension as described under "Stability"). — Sigma.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) from Skeletal Muscle (Rabbit) D-Glyceraldehyde-3-phosphate : NAD oxidoreductase (phosphorylating) *>

D-Glyceraldehyde-3-phosphate + D P N + + P 1,3-diphosphoglycerate + D P N H + H+

G A P D H crystallizes from 2.4 M ammonium sulphate solution (pH 8.5) as rhombic plates superim­

posed on one another. For the isolation of the crystalline enzyme, see e.g.*

1

Molecular weight: 117000; pH optimum: 8.7 in triethanolamine buffer; isoelectric point in 0.1 M phosphate buffer: 6.6; Michaelis constants: 5.1 X 10~5 M for G A P ; 3.9 X 10-5 M for D P N .

Activators, Inhibitors, Chemical Properties:

The prosthetic group of the enzyme is glutathione. A molecule of the enzyme contains 12 to 16 SH groups. Three SH groups can be blocked by heavy metal ions; glutathione or E D T A reverse this inhibition. 10~

4

*) I U B nomenclature

31

32) G. Beisenherz, H. J. Boltze, Th. Biicher, R. Czok, K. H. Garbade, E. Meyer-Arendt and G. Pflei­

derer, Z. Naturforsch. 8b, 555 [1953].

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay o f activity

3 2

Stability:

Suspensions in 2.5 M ( N H ^ S C ^ solution (ca. p H 7.5) are stable for many months at 0 t o 4 ° C . Enzyme solutions or suspensions can be dialysed against 0.001 M N H

4

Purity Required:

A universally suitable G A P D H preparation should contain ca. 25 units/mg. Contaminants: not more than 0.01 % aldolase, glycerol-l-phosphate dehydrogenase, lactic dehydrogenase and pyruvic kinase; 0 . 2 % myokinase; 0 . 0 5 % phosphoglycerate kinase, phosphoglycerate mutase and triose­

phosphate isomerase.

C o m m e r c i a l Preparations:

Boehringer (suspension as described under "Stability"). — Sigma (suspension in (NH

4

tion).

Glycerokinase (GK) from

ATP: glycerol phosphotransferase *)

Glycerol + A T P * glycerol-l-phosphate + A D P

G K crystallizes as fine needles from 2.2 M a m m o n i u m sulphate solution. For the isolation, see e.g.**).

Molecular weight: 2 5 1 0 0 0 ; Michaelis constants: 9 x 1 0

-5

5

Activators, Inhibitors, Chemical Properties:

The enzyme is completely inhibited by 5 x 10~

3

5

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay o f activity

3 4

phate dehydrogenase is the indicator e n z y m e

3 3

Boehringer).

Stability:

The crystalline suspension in 2.2 M (NH

4

ing 0.001 M glycerol and 0.001 M E D T A ) at 0 to 3 ° C without any appreciable loss of activity.

Purity Required:

A universally suitable G K preparation should contain ca. 85 units/mg. Contaminants: not more than 0.01 % glycerol-l-phosphate dehydrogenase, triosephosphate isomerase, myokinase, D P N H oxidase.

Commercial Preparation:

Boehringer (crystalline suspension in 2.2 M ( N H

4

2

*) I U B nomenclature

33) H.-U. Bergmeyer, G. Holz, E. M. Kauder, H. Mollering and O. Wieland, Biochem. Z. 333, 471 [1961].

34) O. Wieland and M. Suyter, Biochem. Z. 329, 320 [1957].

Glycerol-l-phosphate Dehydrogenase (GDH) from Skeletal Muscle (Rabbit) a-Glycerophosphate dehydrogenase; L-glycerol-3-phosphate : NAD oxidoreductase *>

L-Glycerol-1 -phosphate + D P N + ^ ^ dihydroxyacetone phosphate + D P N H + H

+

G D H crystallizes as hexagonal plates from 2.0 M a m m o n i u m sulphate solution (pH 6). For the isolation, see e.g.

35)

Molecular weight: 7 8 0 0 0 ; Michaelis constants: 1.1 x 1 0

-4

-4

for dihydroxyacetone phosphate and 3.8 x 1 0

-4

Activators, Inhibitors, Chemical Properties:

G D H contains ca. 15 SH groups per mole. E D T A activates the enzyme. 1 Mole /?-chloromercuri- benzoate per mole enzyme causes complete inhibition. Fructose-1,6-diphosphate is a competitive inhibitor.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

3 5

Stability:

Crystalline suspensions in 2.0 M ( N H ^ S C ^ solutions (pH 6.0) are stable for years at 0 to 4°C.

Solutions or suspensions can be dialysed against 200 volumes of 1 0

-3

Purity Required:

A universally suitable G D H preparation should contain ca. 35 units/mg. Contaminants: not more than 0.05% tactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, aldolase and pyruvic kinase.

Commercial Preparations:

Boehringer (crystalline suspension as described under "Stability"). — Sigma (crystalline suspension in a m m o n i u m sulphate solution) G D H / T I M , see p. 999.

Glyoxalase I (Gl-I) from Yeast

.S-Lactoyl-glutathione methylglyoxal-lyase (isomerizing) *)

Glutathione + methylglyoxal > .S-lactoyl-glutathione

Gl-I crystallizes from ca. 3.2 M a m m o n i u m sulphate solution (ca. p H 8); the crystalline form has not been described. For the isolation, see e.g.

3 6

Molecular weight has not been determined; Michaelis constant: 7.4 x 1 0

-4

Activators, Inhibitors, Chemical Properties:

N o activators for Gl-I have so far been described. H C N inhibits the enzyme.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

3 6

formation leaflets of various firms, e.g. Boehringer).

*) I U B nomenclature

35) G. Beisenherz, H. J. Boltze, Th. Bucher, R. Czok, K. H. Garbade, E. Meyer-Arendt and G. Pflei- derer, Z. Naturforsch. 8b, 555 [1953].

36) E. Racker, J. biol. Chemistry 190, 685 [1951].

Stability:

Solutions in 3 0 % glycerol are stable for months at 0 to 4°C. Solutions or suspensions can be dialysed against doubly distilled water at 0 to 3 ° C without any significant loss of activity. Suspensions in a m m o n i u m sulphate solution are unstable.

Purity Required:

A universally suitable Gl-I preparation should contain ca. 300 units/mg. Contaminants: not more than 0.01 % glyoxalase II.

Commercial Preparation:

Boehringer (suspension as described under "Stability").

Glyoxylic Acid Reductase (Gly-R) from Spinach Leaves Glycollate: NAD oxidoreductase*)

C H O - C O O H + D P N H + H+ H O H

2

Gly-R crystallizes from ca. 1.2 M ammonium sulphate solution (ca. p H 6); the crystals consist o f fine needles. For the isolation, see e.g.*

1

Molecular weight: the molecular weight has not yet been determined; p H optimum: 6.3 to 6.6;

Michaelis constant: 9.1 x 1 0 ~

3

Activators, Inhibitors, Chemical Properties:

/7-Chloromercuribenzoate, semicarbazide, pyruvate and 2,4-dichlorophenoxyacetate inhibit the enzyme. Semicarbazide probably inhibits by reaction with glyoxylate.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

3 7

Stability:

Crystalline suspensions in 1.5 M ( N H

4

2

Purity Required:

A universally suitable Gly-R preparation should contain ca. 55 units/mg. Contaminants: not more than 0.1 % glycollic acid oxidase, D P N H oxidase and alcohol dehydrogenase.

Commercial Preparation:

Boehringer (suspension as described under "Stability").

Guanase from Rabbit Liver

Guanine deaminase; guanine aminohydrolase*>

Guanine + H

2

3

Guanase has not yet been crystallized. For the isolation of a purified fraction from rat liver, see e.g.*

9

6

for guanine, 7 x 1 0 ~

5

*) I U B nomenclature

37) / . Zelitch, J. biol. Chemistry 276, 553 [19551.

38) H. Holzer and A. Holldorf, Biochem. Z. 329, 292 [1957].

39) H. M. Kalckar, J. biol. Chemistry 167, 461 [1947].

Activators, Inhibitors, Chemical Properties:

Have not yet been described.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

3 9

Stability:

Suspensions in 2.4 M a m m o n i u m sulphate solution (ca. p H 6.5) are stable for at least 6 months at 0 to 4 ° C . Solutions or suspensions of the enzyme can be dialysed against 0.02 M potassium phosphate buffer (pH 7.6) for 6 hours at 0 to 3 ° C with little loss of activity.

Purity Required:

A guanase preparation containing ca. 0.1 units/mg. is suitable for the methods described in this book.

The presence of nucleoside phosphorylase does not interfere. Other contaminants: not more than 1 % adenosine deaminase and alkaline phosphatase.

Commercial Preparation:

Boehringer (suspension as described under "Stability").

Hexokinase (HK) from Yeast ATP: D-hexose-6-phosphotransferase *>

Hexose + A T P

v

H K crystallizes as fine needles from 3.0 M a m m o n i u m sulphate solution (ca. p H 8). For the isolation, see e.g.

40)

Molecular weight: 9 6 6 0 0 ; Michaelis constants: 1.0x 1 0

-4

-7

M g

2+

2+

3

2+

-3

3-

Dichlorodiethylsulphide (mustard gas) causes a complete and irreversible inhibition. Inhibition by c o m p o u n d s which react with SH groups can be reversed by thiols. Glucose protects H K from proteolysis.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity at 340 or 366 mu, with glucose and A T P corresponds in principle to the determination of glucose with H K (see p. 117). In this case G 6 P - D H is the indicator enzyme.

(See the information leaflets o f various firms, e.g. Boehringer).

Stability:

Crystalline suspensions in 3 M ( N H 4 )

2

3

4

*) IUB nomenclature

40

Academic Press, N e w York 1962, Vol. V, p. 226.

Purity Required:

A universally suitable H K preparation should contain ca. 140 units/mg. *). Contaminants: not more than 0 . 6 % phosphoglucose isomerase; 0 . 5 % glutathione reductase; 0 . 0 2 % myokinase; 0 . 0 1 % 6-phosphogluconic dehydrogenase, phosphoglucose mutase and glucose-6-phosphate dehydrogenase.

Commercial Preparations:

Boehringer (suspension as described under "Stability"). — Sigma (5 grades o f dry powder and a suspension as described under "Stability").

(3-Hydroxyacyl-CoA Dehydrogenase (HOADH) from Pig Heart

P-Ketoreductase; (3-ketohydrogenase; L-(+)-(3-oxyacyl dehydrogenase; p-hydroxybutyryl- CoA dehydrogenase; L-3-hydroxyacyl-CoA : NAD oxidoreductase * *>

L-(+)-P-Hydroxybutyryl-CoA + D P N + acetoacetyl-CoA + D P N H + H+

H O A D H crystallizes as fine needles from 2.8 M ammonium sulphate solution. For the isolation, see e.g.

41

42

Molecular weight: not yet determined exactly; p H optimum for the oxidation: 9.6; Michaelis con­

stants: 1 0 ~

2

_ 5

10~

4

Equilibrium constant:

=

+3 =

[acetoacetyl-CoA] [ D P N H ] [H+]

Activators, Inhibitors, Chemical Properties:

1 0

-4

-3

£

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay o f activity

4 3

Stability:

The crystalline suspension in 2.8 M (NH4)

2

KHCO3

Purity Required:

A universally suitable H O A D H preparation should contain ca. 9 units/mg. Contaminants: not more than 1 % malic dehydrogenase; 0 . 0 1 % crotonase, a-oxoglutarate dehydrogenase and butyryl-CoA dehydrogenase.

Commercial Preparations:

Boehringer (crystalline suspension as described under "Stability").

*) For conversion to other units, see p. 545.

**) I U B nomenclature

4

4

43

Invertase from Yeast

Saccharase; sucrase; (3-fructofuranosidase; p(h)-fructosidase; P-D-fructofuranoside fructohydrolase *>

Sucrose + H2O > glucose + fructose Invertase has not been crystallized. For the isolation, see e.g.

44

Molecular weight: the published values for the molecular weight vary between 6 0 0 0 0 and 123000;

Michaelis constants: 1.6 x 10~

2

_1

Activators, Inhibitors, Chemical Properties:

N o activators are known. Fluoride does not inhibit. Heavy metals such as C u

2 +

2+

+

cause a reversible inhibition; it can be reversed by dialysis or by chelating agents. Carbonyl reagents, such as aniline, /?-toluidine and phenylhydrazine inhibit. Saccharides, glycerol and tartrate stabilize the enzyme.

D e t e r m i n a t i o n of A c t i v i t y :

Iodometric or polarimetric assay of activity after hydrolysis of sucrose (see p. 901) or the spectro­

photometric enzymatic assay of the glucose or fructose liberated (see p. 117, 156).

Stability:

Dry powders or stabilized solutions (see above) are stable for months at 0 to 4° C. Solutions can be dialysed against running water to remove the stabilizer.

Purity Required:

A universally suitable invertase preparation should contain ca. 500 units **Vmg. The preparation must be free from melibiase, maltase, (3-galactosidase and a-glucosidase.

Commercial P r e p a r a t i o n s :

California Corporation (dry powder). — E. Merck, Darmstadt (Germany) (solution). — Dr. Th.

Schuchardt G m b H . , Munich (Germany) (solution). — Fluka, Buchs, St. Gallen (Switzerland) (solution).

Isocitric Dehydrogenase (ICDH) from Pig Heart (TPN-specific) L s -Isocitrate : NADP oxidoreductase (decarboxylating) *)

L

s

2

I C D H has not yet been crystallized. For the isolation of an extensively purified preparation, see Molecular weight: 6 1 0 0 0 ; isoelectric point: 7.4; p H o p t i m u m : p H 7.4; Equilibrium constant at p H 7 and 22° C:

_ [L

S

K

2

= 3 [ L / m 1

l e ]

Michaelis constant: 2.6 x 10~6 M for L

s

*) I U B nomenclature

**) One unit is the amount of enzyme which converts 1 u,mole of substrate in 1 min. (refer to p. 32), For an example of an assay mixture, see p. 902 under enzymatic reaction.

44

4

Activators, Inhibitors, C h e m i c a l Properties:

The optimal M n

2+

-4

-3

2+

2.5 x 1 0

-8

5

Very small amounts of Cu or H g c o m p o u n d s cause complete inhibition and this is reversed by m o n o - thiols. The decarboxylation of oxalosuccinate is inhibited by an excess of isocitrate.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

4 6

s

formation leaflets o f various firms, e.g. Boehringer).

Stability:

Dry preparations, suspensions in 3.0 M ( N H ^ S C ^ solution (ca. p H 6.0) and Solutions in 5 0 % glycerol are stable for many months at 0 to 4 ° C . Enzyme solutions can be dialysed for short periods only. Dialysis for t w o hours against 0.01 M phosphate buffer (pH 5.8) + 0.001 M E D T A at 2 ° C causes a ca. 2 0 % loss of activity.

Purity R e q u i r e d :

A universally suitable I C D H preparation should contain ca. 2 units/mg. Contaminants: not more than 0 . 2 % aconitase and DPN-specific I C D H .

C o m m e r c i a l Preparations:

Boehringer (glycerol solution as described under "Stability"). — Sigma (Type I, crude, TPN-specific).

Lactic Dehydrogenase (LDH) from Skeletal Muscle (Rabbit) L-Lactate: NAD oxidoreductase *)

L - ( + ) - L a c t a t e + D P N + pyruvate + D P N H + H+

L D H crystallizes as very fine needles from 2.1 a m m o n i u m sulphate solution. For the isolation, see e.g«l

Molecular weight: ca. 120000; Michaelis constants: 5 x 1 0 ~

6

5

optimum conditions: at 39°C, p H 7.4 and 3 x 10~

3

A c t i v a t o r s , Inhibitors, C h e m i c a l Properties:

Whether L D H contains zinc is still in dispute. 1 Molecule L D H contains 3 molecules D P N and ca.

10 S H groups. The enzyme forms an addition c o m p o u n d with sulphite and D P N , which is relatively stable to heat denaturation, the action of urea, proteolysis, p-chloromercuribenzoate or dinitro- fluorobenzene. Iodoacetate and 7V~-ethyrmaleinimide have no action o n the enzyme. Oxamate and oxalate form inactive complexes with L D H . Pyridine-3-sulphonate is a competitive inhibitor.

D e t e r m i n a t i o n of A c t i v i t y :

Spectrophotometric assay of activity

4 7

tion leaflets of various firms, e.g. Boehringer and Worthington).

Stability:

Very dilute solutions are unstable, while crystalline suspensions in ( N H

4

2

4

3

*) I U B nomenclature

46

47