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Direct and continuous spectrophotometric assay of phosphomonoesterases
Direct and Continuous Spectrophotometric Phosphomonoesterases
Assay of
B. H. J. Hofstee FWIIL
ihc
Palo
Alto
Medical
Reseurch
Foundation,
Received Kwember
I’trlo
.1 lto,
(‘ctlijornia
10.1953
In contrast to routine activity det’erminat8ionsunder standard conditions, kinetic studies of an enzyme system often require analysis of the reaction mixture at frequent time intervals, which can be accomplished more readily with a method that allows direct, and continuous determination of the progress of the reaction. In previous communications (1, 2) such a method has been described for esterasesthat catalyze the hydrolysis of n-fatty acid esters of hydroxybenzoic acids. It is based on the fact &at hydroxybenzoic acids strongly absorb light of a wavelengt,h of aromld 300 rnp, while the esters do not absorb at all in this region. The method allows determination of initial reaction rates over a 10~500-fold range of the substrate conccntration and permits detection of as little as 0.01 pmole of free hydroxy; benzoic acid. Measurements can be made wit,h relatively impure enzyme preparations. Existing photometric and sensitive methods for the determination of phosphomonoesteraseact’ivity, such as those in which glycerophosphoric acids (3, 4), phenolphthalein phosphate (5) phenyl phosphate (6), pnitrophenyl phosphate (7, 8) or fl-naphthyl phosphate (9) are used as the substrate, either cannot easily be adapted to direct and continuous determinat’ions or can only be used for det’erminations in a limited p1-I range. In the present paper it will be shown that with phosphorylmonosalicylic acid as the substrate, the above principle can be applied to a sensitive, accurate, direct and continuous method for the determination of “acid” as well as “alkaline” phosphatase. 139
140
B. H. J. HOFSTEE EXPERIMENTAL
The Substrate Preparation. PhosphorylmonosaIicylie acid can be prepared salicylic acid and phosphorus pentachloride in a mole-to-mole treatment of the product with water (10-13). Anschtitz (12) found the reaction to proceed as follows:
COOH.CeH,.OH COCI.GHI.OPOC~~
+ PCl~+COCI~C~H~~OPOCl~ + 3HzO+COOH.CsH~OPO(OH)z
by simply mixing ratio followed by
+ 2HCl + 3HCl
Onlythe primary ester, essentially free of secondary and t,ertiary esters, appeared to be formed. The melting point of a preparation that still contained some impurity was 147”. No satisfactory procedure for recrystallization is given. On the basis of this and other (14) information, the following procedure has been worked out. SalicyIic acid is added to phosphorus pentachloride in a mole-to-mole ratio. The pentachloride is suspended in toluene by constant agitation. After the salicylic acid has been added in small portions, a clear and colorless solution is obtained, which is exposed for several days to water vapor. For this purpose the solution is placed in a flat dish in a closed jar containing a small amount of water. The ester crystallizes out, is removed by suction, and is washed with toluene. The compound is recrystallized several times from a solution in warm dioxane to which toluene or petroleum ether is added until a turbidity is barely visible. In the cold room it then slowly crystallizes in clusters of macroscopic needles. Yield: 50-60% of theory.
Assay. For the determination of its salicylic and phosphoric acid content, the compound was hydrolyzed in acetate buffer pH 5 (seeProperties). Hydrolysis of a 10B3M solution was complete in about an hour at 80-90”. The salicylic acid was determined by direct spectrophotometric measurement (Fig. l), and for phosphate the method of Fiske and SubbaRow (15) was used. The results, based on a molecular weight of 218 (no water of crystallization), are given in the following tabulation, in which also other criteria of purity are recorded. Salicylic
acid %
Theoretical Found
63.3 62.2
Phosphorus %
14.2 14.1
Number of acid equivalents
3.0 3.0
Carbon %
38.53 38.48
Hydrogen %
3.21 3.41
Properties. The compound occurs as a white crystalline nonhygroscopic powder. It is extremely solubIe in water and alcohol, very soluble in dioxane, not soluble in petroleum ether or toluene. The melting point is 154-155”.
ASSAY
0
OF
2
141
PHOSPHOMONOESTERASES
4
6
8
IO
12
PH 1. The influence of pH on the light of phosphorylmonosalicylic acid.
FIG.
and
absorption
(310
mr)
of salicylic
acid
h titration curve (Fig. 2, part A) reveals pK values of about 1.9, 3.8, and 6.5. In Fig. 2, part B, the influence of pH on the rat,e of spontaneous hydrolysis of a 3.3 X 1OV M solution is given. The curve shows a maximum at pH 5.2. This maximum coincides with the pH at which two of the three acid groups have been neutralized. On the other hand, after complete neutralizat,ion the compound is very stable. The compound is also stable in acid solutions (pH < 2), although to a lesser ext,ent. These findings are similar to those of Desjobert (l(i), who reports that the pH of a maximum of hydrolysis of a primary ester such as monoethylort,hophosphoric acid corresponds to the point of half neutralization . The absorption spectrum of the phosphoryl ester is similar to that of t,he n-fatty acid esters of salicylic acid (1) but is shifted somewhat to higher wavelengths and therefore shows a slight absorption in the region (205-300 mp) where salicylic acid displays a maximum. Enzyme Preparations (a) A 5% centrifuged (b) A 1% centrifuged
homogenate homogenate
(Waring blendor) of rat liver of rat kidney in water.
in wat,er
142
B.
H.
IVIY
J.
HOFSTEE
““ITVC
I :
i I
/
: It
I
I I
: ,
I
i II:
‘1
; I
; NON-ENiZYM
HYDdOLY&S 30 i min. at
A
PH Fro. 2. Part 8. Titration curve of phosphorylmonosalicylic acid. Part influence of pH on the non-enzymic hydrolysis of phosphorylmonosalicylic
B.
The
acid.
(c) A 1% centrifuged aqueous extract of calf duodenum powder. The powder was provided by courtesy of Vio Bin Corporation, Monticello, Illinois. Activity
The activity
Determinations
(initial reaction rate) of the different enzyme preparations
was determined by direct measurement, at frequent time intervals, of the increase in extinction of a mixture of 0.1 ml. of the enzyme preparation,
substrate, and buffer (total volume 3 ml.) over a 15-30-min. period. The amount. of substrate hydrolyzed was derived from the data in Fig. 1. The titration curve (Fig. 2, part, A) showsthat the substrate possesses buffering capacity over a pH range from 2.5 to 7.5 although at a pH of about 5 this capacity is rather weak. For this reason acetic acid was included in the buffer-substrate solutions. Buffering in the region pH
ASSAY
OF
143
PHOSPHOMONOESTERASES
7.5-10.5 was provided by glycylglycine (pK 8.0) and glycine (pK 0.8). The extinction was measured at 310 rnp in a Beckman spectrophotometer, model DU, which was provided with a thermospacer through which water of 30” was circulated. Measuremellt at 310 rnp has the advantage of low absorption by the substrate, which reduces interference with activity determination at high substrate concentrations. h further advantage of measurement at this wavelength, despite a small sacrifice in sensitivity, is that the absorption of proteins is low, which is convenient in work with crude enzyme preparations. The contents of the reference cuvette were identical with t’hose of the reaction mixture except that it contained no enzyme. This procedure canceled automatically the influence of non-enzymic hydrolysis on the observed increase in extinction of the reaction mixture. When using fresh tissue extracts, reaction mixtures of low pH were oftell slightly turbid or opalescent. However, changes in extinction of
PHOSPHO-MONO-ESTERASES IN TISSUES
2
3
4
5
6 PH
7
8
9
IO
Fra. 3. The influence of pH on the phosphomonoesterase activities of rat liver, of rat kidney, and of calf duodenum. The different characters indicate different experiments. Substrate: phosphorylmonosalicylic acid, 3.3 X 10-z M. Buffer: mixture of substrate, acetate, glycylglycine and glycine, each 3.3 X
10-M.
144
B.
H.
J.
HOFSTEE
the reaction mixture minus the substrate, as well as changes due to non-enzymic interaction of the substrate with the heated enzyme (enzyme blank), were negligible whenever checked. When necessary they can be accounted for by inserting a cuvette without substrate and a cuvette with substrate and heated enzyme, respectively. In most cases constant reaction rate was observed over a period of more than 15 min., while reasonable proportionality between enzyme concentration and reaction rate was obtained. Applicability,
Sensitivity
and Accuracy
of the Method
Figure 3 indicates that “acid” as well as “alkaline” phosphatase activity can be determined with the method. Table I shows that the pH optima of 3.545, 5-6, and 9-10 that were obtained, correspond to those of phosphomonoesterases that are known to occur in the respective tissues. The influence of Mg++ is similar as well. Without addition of activators, with a substrate concentration of 3.3 X 1OP M and at the pH optima, amounts of wet tissue of the order of 1 mg. hydrolyze about 0.2 pmoles of substrate per 30 min. at 30”. Depending on the pH, this corresponds to a AE of 0.13-0.20 (Fig. l), which can be read accurately in the spectrophotometer. A further indication of the sensitivity of the method is that in the reaction mixture the enzyme concentration may be of the order of 3000 times as low as in the natural tissues. It can further be pointed out that the activities as recorded in Fig. 3 were not determined under optimal conditions. It was found that with exception of the phosphatase in calf duodenum the enzymes were not saturated with substrate at the given concentration. In neither case was Mg++ added as an activator. Since the only purpose TABLE Comparison
Enzyme
of Properties
source
Literature
I
of Phosphatases Attacking Phosphorylmonosalicylic with Those of Known Phosphatases pH optimum (17)
Influence Found
Literature
(17)
of Mg++ Found (10”
Acid
Y Mg++)
Liver 3.4-4.2 =4 Inhibition =lO% inhibition 5.0-5.5 =5.5 None None Liver 8.6-9.4 8.9 Activation -10% activation Kidney Duodenum 9.8” -100% activation Q A pH optimum of 10.0-10.1 has been reported (8) for alkaline phosphatase human blood serum.
in
ASSSY
OF
145
PHOSPHOMONOESTERASES
was to show the applicability of the proposed met,hod and t’o ident#ify the respective phosphatases, detailed kinetic data w-ill not) be given in the present paper. Subsequent communications will deal with the tlctermination of kinetic constants, and the influence of pH and of added substances on these constants. The accuracy of the method depends on the experiment’al conditions, but may be as high as 100 f 1%. In general, the comments that have been made (2) on the accuracy of the det’erminat,ioll of activity of ot,her &erases wit,h this method also apply to phosphatasr d&trminatioll. SUMMAIZY
A spectrophotometric method has been described for direct and continuous measurement of the activity of phosphomonoestBerases over t,hc pH range of 2-11. At 310 rnp, salicylic acid (high absorptioll) is measured directly in the reaction mixture as reaction product. of the substrate phosphorylmonosalicylic acid (low a,bsorpt,ion). Pnder optimal condtions the activity of “acid” as well as “alkaline” phosphatase can bc determined in crude extracts corresponding to amounts of wet, tissue as low as 1 mg. A det,ailed description of the preparation and propert,& of thtb substrate has been given. NOTE
ADDED
IN
PROOF
While this paper was in press, it came to our :Lttention and Hanson (18) h:tve also used the present suhstr:lte for phosphomonoester~ses.
th:tt Brandenbergel the detwminxtion
of
REFERENCES 1. HOFSTEE, 2.
3. 4. 5. 6. 7. 8. 0.
10. 11. 12.
B. H. J., Science 114, 128 (1951). B. H. J., J. Biol. Chem. 199, 357 (1952). KAY, FI. Il., J. Rio!. C/tern. 89, 235, 249 (1930). I%OD.MSKY, A., J. Biol. Chem. 101, 93 (1933). HUGGINS, C., ANU TALALAY, P., J. Biol. Chem. 169, 399 (1945). KING, E. J., AND ARMSTRONG, R., Can. Med. ksoc. .I. 31, 376 (1934). OHMORI, Y., Enzynaologia 4, 217 (1937). BESSEY, 0. A., LO~YRY, 0. II., BND BROCK, M. J., J. Biol. Chern. 164,321 SELIGMAN, A. M., CHAUNCEY, H. H., KACMLAS, M. M., MANNHEIMER, AND RAVIN, H. A., J. Biol. Chem. 190, 7 (1951). COUPER, RI., Compt. rend. 46, 1107 (1858). CHASANOWITCH, J., Ber. 20, 1165 (1887). AKSCII~~TZ, R., Ann. 228, 308 (1885). HOFSTEE,
(1946) I,.
IT.,
146
B.H.
J. HOFSTEE
13. ANSCH~~TZ, R. AND MOORE, G. D., Ann. 239,314 (1887). 14. KOSOLAPOFF, G. M., “Organophosphorus Compounds.” John Wiley & Sons, Inc., New York, 1950. 15. FISKE, C. H., AND SUBBAROW, Y., J. BioE. Chem. 66,375 (1925). 16. DESJOBERT, M. A., Bull. sot. chim. 11-12, 809 (1947). 17. ROCHH, J., in “The Enzymes,” J. B. SUMNER AND K. MYRBLCK (eds.), Vol. I, p. 481. Academic Press, New York, 1950. 18. BRANDENBERGER, H., AND HANSON, R., Helv. Chim. Acta 36, 900 (1953).
Assay of
B. H. J. Hofstee FWIIL
ihc
Palo
Alto
Medical
Reseurch
Foundation,
Received Kwember
I’trlo
.1 lto,
(‘ctlijornia
10.1953
In contrast to routine activity det’erminat8ionsunder standard conditions, kinetic studies of an enzyme system often require analysis of the reaction mixture at frequent time intervals, which can be accomplished more readily with a method that allows direct, and continuous determination of the progress of the reaction. In previous communications (1, 2) such a method has been described for esterasesthat catalyze the hydrolysis of n-fatty acid esters of hydroxybenzoic acids. It is based on the fact &at hydroxybenzoic acids strongly absorb light of a wavelengt,h of aromld 300 rnp, while the esters do not absorb at all in this region. The method allows determination of initial reaction rates over a 10~500-fold range of the substrate conccntration and permits detection of as little as 0.01 pmole of free hydroxy; benzoic acid. Measurements can be made wit,h relatively impure enzyme preparations. Existing photometric and sensitive methods for the determination of phosphomonoesteraseact’ivity, such as those in which glycerophosphoric acids (3, 4), phenolphthalein phosphate (5) phenyl phosphate (6), pnitrophenyl phosphate (7, 8) or fl-naphthyl phosphate (9) are used as the substrate, either cannot easily be adapted to direct and continuous determinat’ions or can only be used for det’erminations in a limited p1-I range. In the present paper it will be shown that with phosphorylmonosalicylic acid as the substrate, the above principle can be applied to a sensitive, accurate, direct and continuous method for the determination of “acid” as well as “alkaline” phosphatase. 139
140
B. H. J. HOFSTEE EXPERIMENTAL
The Substrate Preparation. PhosphorylmonosaIicylie acid can be prepared salicylic acid and phosphorus pentachloride in a mole-to-mole treatment of the product with water (10-13). Anschtitz (12) found the reaction to proceed as follows:
COOH.CeH,.OH COCI.GHI.OPOC~~
+ PCl~+COCI~C~H~~OPOCl~ + 3HzO+COOH.CsH~OPO(OH)z
by simply mixing ratio followed by
+ 2HCl + 3HCl
Onlythe primary ester, essentially free of secondary and t,ertiary esters, appeared to be formed. The melting point of a preparation that still contained some impurity was 147”. No satisfactory procedure for recrystallization is given. On the basis of this and other (14) information, the following procedure has been worked out. SalicyIic acid is added to phosphorus pentachloride in a mole-to-mole ratio. The pentachloride is suspended in toluene by constant agitation. After the salicylic acid has been added in small portions, a clear and colorless solution is obtained, which is exposed for several days to water vapor. For this purpose the solution is placed in a flat dish in a closed jar containing a small amount of water. The ester crystallizes out, is removed by suction, and is washed with toluene. The compound is recrystallized several times from a solution in warm dioxane to which toluene or petroleum ether is added until a turbidity is barely visible. In the cold room it then slowly crystallizes in clusters of macroscopic needles. Yield: 50-60% of theory.
Assay. For the determination of its salicylic and phosphoric acid content, the compound was hydrolyzed in acetate buffer pH 5 (seeProperties). Hydrolysis of a 10B3M solution was complete in about an hour at 80-90”. The salicylic acid was determined by direct spectrophotometric measurement (Fig. l), and for phosphate the method of Fiske and SubbaRow (15) was used. The results, based on a molecular weight of 218 (no water of crystallization), are given in the following tabulation, in which also other criteria of purity are recorded. Salicylic
acid %
Theoretical Found
63.3 62.2
Phosphorus %
14.2 14.1
Number of acid equivalents
3.0 3.0
Carbon %
38.53 38.48
Hydrogen %
3.21 3.41
Properties. The compound occurs as a white crystalline nonhygroscopic powder. It is extremely solubIe in water and alcohol, very soluble in dioxane, not soluble in petroleum ether or toluene. The melting point is 154-155”.
ASSAY
0
OF
2
141
PHOSPHOMONOESTERASES
4
6
8
IO
12
PH 1. The influence of pH on the light of phosphorylmonosalicylic acid.
FIG.
and
absorption
(310
mr)
of salicylic
acid
h titration curve (Fig. 2, part A) reveals pK values of about 1.9, 3.8, and 6.5. In Fig. 2, part B, the influence of pH on the rat,e of spontaneous hydrolysis of a 3.3 X 1OV M solution is given. The curve shows a maximum at pH 5.2. This maximum coincides with the pH at which two of the three acid groups have been neutralized. On the other hand, after complete neutralizat,ion the compound is very stable. The compound is also stable in acid solutions (pH < 2), although to a lesser ext,ent. These findings are similar to those of Desjobert (l(i), who reports that the pH of a maximum of hydrolysis of a primary ester such as monoethylort,hophosphoric acid corresponds to the point of half neutralization . The absorption spectrum of the phosphoryl ester is similar to that of t,he n-fatty acid esters of salicylic acid (1) but is shifted somewhat to higher wavelengths and therefore shows a slight absorption in the region (205-300 mp) where salicylic acid displays a maximum. Enzyme Preparations (a) A 5% centrifuged (b) A 1% centrifuged
homogenate homogenate
(Waring blendor) of rat liver of rat kidney in water.
in wat,er
142
B.
H.
IVIY
J.
HOFSTEE
““ITVC
I :
i I
/
: It
I
I I
: ,
I
i II:
‘1
; I
; NON-ENiZYM
HYDdOLY&S 30 i min. at
A
PH Fro. 2. Part 8. Titration curve of phosphorylmonosalicylic acid. Part influence of pH on the non-enzymic hydrolysis of phosphorylmonosalicylic
B.
The
acid.
(c) A 1% centrifuged aqueous extract of calf duodenum powder. The powder was provided by courtesy of Vio Bin Corporation, Monticello, Illinois. Activity
The activity
Determinations
(initial reaction rate) of the different enzyme preparations
was determined by direct measurement, at frequent time intervals, of the increase in extinction of a mixture of 0.1 ml. of the enzyme preparation,
substrate, and buffer (total volume 3 ml.) over a 15-30-min. period. The amount. of substrate hydrolyzed was derived from the data in Fig. 1. The titration curve (Fig. 2, part, A) showsthat the substrate possesses buffering capacity over a pH range from 2.5 to 7.5 although at a pH of about 5 this capacity is rather weak. For this reason acetic acid was included in the buffer-substrate solutions. Buffering in the region pH
ASSAY
OF
143
PHOSPHOMONOESTERASES
7.5-10.5 was provided by glycylglycine (pK 8.0) and glycine (pK 0.8). The extinction was measured at 310 rnp in a Beckman spectrophotometer, model DU, which was provided with a thermospacer through which water of 30” was circulated. Measuremellt at 310 rnp has the advantage of low absorption by the substrate, which reduces interference with activity determination at high substrate concentrations. h further advantage of measurement at this wavelength, despite a small sacrifice in sensitivity, is that the absorption of proteins is low, which is convenient in work with crude enzyme preparations. The contents of the reference cuvette were identical with t’hose of the reaction mixture except that it contained no enzyme. This procedure canceled automatically the influence of non-enzymic hydrolysis on the observed increase in extinction of the reaction mixture. When using fresh tissue extracts, reaction mixtures of low pH were oftell slightly turbid or opalescent. However, changes in extinction of
PHOSPHO-MONO-ESTERASES IN TISSUES
2
3
4
5
6 PH
7
8
9
IO
Fra. 3. The influence of pH on the phosphomonoesterase activities of rat liver, of rat kidney, and of calf duodenum. The different characters indicate different experiments. Substrate: phosphorylmonosalicylic acid, 3.3 X 10-z M. Buffer: mixture of substrate, acetate, glycylglycine and glycine, each 3.3 X
10-M.
144
B.
H.
J.
HOFSTEE
the reaction mixture minus the substrate, as well as changes due to non-enzymic interaction of the substrate with the heated enzyme (enzyme blank), were negligible whenever checked. When necessary they can be accounted for by inserting a cuvette without substrate and a cuvette with substrate and heated enzyme, respectively. In most cases constant reaction rate was observed over a period of more than 15 min., while reasonable proportionality between enzyme concentration and reaction rate was obtained. Applicability,
Sensitivity
and Accuracy
of the Method
Figure 3 indicates that “acid” as well as “alkaline” phosphatase activity can be determined with the method. Table I shows that the pH optima of 3.545, 5-6, and 9-10 that were obtained, correspond to those of phosphomonoesterases that are known to occur in the respective tissues. The influence of Mg++ is similar as well. Without addition of activators, with a substrate concentration of 3.3 X 1OP M and at the pH optima, amounts of wet tissue of the order of 1 mg. hydrolyze about 0.2 pmoles of substrate per 30 min. at 30”. Depending on the pH, this corresponds to a AE of 0.13-0.20 (Fig. l), which can be read accurately in the spectrophotometer. A further indication of the sensitivity of the method is that in the reaction mixture the enzyme concentration may be of the order of 3000 times as low as in the natural tissues. It can further be pointed out that the activities as recorded in Fig. 3 were not determined under optimal conditions. It was found that with exception of the phosphatase in calf duodenum the enzymes were not saturated with substrate at the given concentration. In neither case was Mg++ added as an activator. Since the only purpose TABLE Comparison
Enzyme
of Properties
source
Literature
I
of Phosphatases Attacking Phosphorylmonosalicylic with Those of Known Phosphatases pH optimum (17)
Influence Found
Literature
(17)
of Mg++ Found (10”
Acid
Y Mg++)
Liver 3.4-4.2 =4 Inhibition =lO% inhibition 5.0-5.5 =5.5 None None Liver 8.6-9.4 8.9 Activation -10% activation Kidney Duodenum 9.8” -100% activation Q A pH optimum of 10.0-10.1 has been reported (8) for alkaline phosphatase human blood serum.
in
ASSSY
OF
145
PHOSPHOMONOESTERASES
was to show the applicability of the proposed met,hod and t’o ident#ify the respective phosphatases, detailed kinetic data w-ill not) be given in the present paper. Subsequent communications will deal with the tlctermination of kinetic constants, and the influence of pH and of added substances on these constants. The accuracy of the method depends on the experiment’al conditions, but may be as high as 100 f 1%. In general, the comments that have been made (2) on the accuracy of the det’erminat,ioll of activity of ot,her &erases wit,h this method also apply to phosphatasr d&trminatioll. SUMMAIZY
A spectrophotometric method has been described for direct and continuous measurement of the activity of phosphomonoestBerases over t,hc pH range of 2-11. At 310 rnp, salicylic acid (high absorptioll) is measured directly in the reaction mixture as reaction product. of the substrate phosphorylmonosalicylic acid (low a,bsorpt,ion). Pnder optimal condtions the activity of “acid” as well as “alkaline” phosphatase can bc determined in crude extracts corresponding to amounts of wet, tissue as low as 1 mg. A det,ailed description of the preparation and propert,& of thtb substrate has been given. NOTE
ADDED
IN
PROOF
While this paper was in press, it came to our :Lttention and Hanson (18) h:tve also used the present suhstr:lte for phosphomonoester~ses.
th:tt Brandenbergel the detwminxtion
of
REFERENCES 1. HOFSTEE, 2.
3. 4. 5. 6. 7. 8. 0.
10. 11. 12.
B. H. J., Science 114, 128 (1951). B. H. J., J. Biol. Chem. 199, 357 (1952). KAY, FI. Il., J. Rio!. C/tern. 89, 235, 249 (1930). I%OD.MSKY, A., J. Biol. Chem. 101, 93 (1933). HUGGINS, C., ANU TALALAY, P., J. Biol. Chem. 169, 399 (1945). KING, E. J., AND ARMSTRONG, R., Can. Med. ksoc. .I. 31, 376 (1934). OHMORI, Y., Enzynaologia 4, 217 (1937). BESSEY, 0. A., LO~YRY, 0. II., BND BROCK, M. J., J. Biol. Chern. 164,321 SELIGMAN, A. M., CHAUNCEY, H. H., KACMLAS, M. M., MANNHEIMER, AND RAVIN, H. A., J. Biol. Chem. 190, 7 (1951). COUPER, RI., Compt. rend. 46, 1107 (1858). CHASANOWITCH, J., Ber. 20, 1165 (1887). AKSCII~~TZ, R., Ann. 228, 308 (1885). HOFSTEE,
(1946) I,.
IT.,
146
B.H.
J. HOFSTEE
13. ANSCH~~TZ, R. AND MOORE, G. D., Ann. 239,314 (1887). 14. KOSOLAPOFF, G. M., “Organophosphorus Compounds.” John Wiley & Sons, Inc., New York, 1950. 15. FISKE, C. H., AND SUBBAROW, Y., J. BioE. Chem. 66,375 (1925). 16. DESJOBERT, M. A., Bull. sot. chim. 11-12, 809 (1947). 17. ROCHH, J., in “The Enzymes,” J. B. SUMNER AND K. MYRBLCK (eds.), Vol. I, p. 481. Academic Press, New York, 1950. 18. BRANDENBERGER, H., AND HANSON, R., Helv. Chim. Acta 36, 900 (1953).