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Studies on glutamic-oxalacetic transaminase. II. The properties of two derivatives of pyridoxal 5-phosphate
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
300-309 (1959)
82,
Studies on Glutamic-Oxalacetic Transaminase. II. The Properties of Two Derivatives of Pyridoxal 5-Phosphate Vincenzo Bonavita and Vincenzo Scardi From
the Laboratory
of Human
Physiology,
Received
October
University
oj Ferrara,
Ferrara,
Italy
2, 1958
INTRODUCTION
A complete study on the properties of the isonicotinoylhydrazone and the cyanohydrin of pyridoxal Sphosphate (Py5P) has been undertaken in order to elucidate the mechanism of the interaction between Py5P and its apoenzymes (l-3). The present paper deals with results on the ionizable groups of both compounds, as compared with the native coenzyme, and on their spectrofluorometric properties. EXPERIMENTAL Pyridoxal 5-phosphate (Hoffmann-LaRoche) and isonieotinic acid hydrazide Lepetit (Nicotibina Lepetit, in crystals) have been used. The isonicotinoylhydrazone of Py5P (PyBPINH) has been prepared by direct reaction of stoichiometric amounts of both reactants, following the method suggested by Sah (4) for thechemical synthesis of the isonicotinoylhydraaone of pyridoxal hydrochloride. The yield was 6@64oj The reaction between Py5P and isoniazid (INH) in sodium phosphate buffer 0.05 M at different pH values has been followed as well in the experiments of Davison (5), in a Beckman spectrophotometer, model DU, equipped with a thermospacer. Figure 1 shows the rate of formation of Py5PINH with excess INH at 17” C., at pH values ranging from 5.0 s 8.0. It is evident that the rate is higher at the lower pH. The activation energy was found equal to 8700 cal./mole. This value does not agree with the value found by Davison (5) (14,000 cal./mole). The melting point is >230” C. And. Calcd. for C~~HISOBN~P: C, 46; H, 4.1; N, 15.3; P, 8.45. Found: C, 44.87; H, 4.23; N, 15.05; P, 7.95. The cyanohydrin of Py5P (Py5PCN) has been prepared by us as previously described (1, 6). The absorption spectra have been measured at room temperature (18” C.) in a Beckman spectrophotometer, model DU. Fluorescence spectra have been taken with a Farrand spectrofluorometer. Absorption and fluorescence spectra were measured at intervals of 0.4 pH unit, between 1 and 11, in order to obtain spectrophotometrically evident pK,’ values. pH measurements were made with a Beckman potentiometer. 300
DERIVATIVES
301
OF PYRIDOXAL &PHOSPHATE RESULTS
Absorption Spectra at Diferent
pH Values
Figure 2 shows the absorption spectra of Py5PINH at pH’s 3 and 5.2. The extinction maximum shifts from 300 rnp at pH 3 to 290 rnp at pH 5.2. The difference, Es40 - EzsO, was chosen for the calculation of this pK,‘. Figure 3 shows the absorption spectra of the same compound at pH’s 7 and 9. A shift of the extinction maximum from 292 to 310 rnp is evident. The absorption spectrum at pH 9 shows an almost complete fusion of the bands which at neutral pH have distinct maxima at 292 and at 340 mp. The difference, Em - Es30 , was chosen for the pK,’ calculation.
E292
.700
so0
.3oc
.lOC
5
10
FIG. 1. Rat,e of interaction between Py5P and INH at different 5 (0), pH 6 (O), pH 7 (o), pH 8 (X). INN excess: 25 times.
min.
pH values: pH
302
BONAVITA
AND
SCARD~
Figure 4 shows the absorption spectra of Py5PINH in the range 200280 mr, at pH’s 1 and 3. The difference, Ez6” - A&, , was chosen for this calculation. The pK,’ value was also confirmed from measurement of E 330 - E340 . In the former instance a pK,’ of 2.75, in the latter of 2.80, was found. Figure 5 shows the absorption spectra of Py5PINH between 200 and 280 rnp, at pH’s 5.4 and 7.4. The difference, Es,, - Ezo3, was chosen for the calculation. Figure 6 shows the absorption spectra of Py5PCN at pH’s 5 and 6.6. The difference, E2To- Ezoo, was chosen for the pK,’ calculation. Figure 7 shows the absorption spectra of the same compound at pH’a 9.1 and 11.1, with a shift of the extinction maximum from 318 to 308 mp. The difference, E320- E310, was chosen.
E 1.300
1.100
.900
.700
so0
,300
300 250 FIG. 2. Absorption spectra pH 3, .- - -0 pH 5.2.
300 of Py5PINH,
400
350 W4
M, in
acetate
m)r
buffer,
0.1
M: O--O
DERIVATIVES
OF
PYRIDOXAL
303
&PHOSPHATE
Fluorescence Spectra at Diflerent pH Values Fluorescence spectra of Py5P and Py5PINH have shown a great similarity. Both compounds, however, are not highly fluorescent in the pH range l-11. Py5PCN shows a fluorescence spectrum of peculiar interest, which has permitted the measurement of a pK,’ value around 5.85, very similar to that derived from the absorption specka (5.93). Figure 8 shows the fluorescence spectra of Py5PCN at pH’s 5 and 6.2. This quenching from neutralization has permitted us to define as highly fluorescent the form of the cyanohydrin having the phenolic hydroxyl group uncharged.
250 FIG.
pH7,
3. Absorption l --- l pH9.
300
spectra
350
of PyBPINH,
400
w
1W’ M, in tris-HCl,
0.1 M:
O--O
304
BONAVITA
AND
SCARDI
Figure 9 shows the fluorescence spectra of Py5PCN at pH’s 1 and 3. There is a clear quenching from excessive hydrogen-ion concentration, which has permitted us to measure a pK,’ < 2.50, evidently due to the dissociation of the primary phosphate group. This pK,’ is not evident from the absorption spectra. The fluorescence spectra at higher pH values have shown a gradual quenching but it was not possible to measure further pK,’ values. E .soo
.800
.600
. 500
.400
.300
.200
*loo
250
200
4. Absorption spectra M:O--OpHl, l ---•pH3. FIG.
of Py5PINH,
0.5 X 10-”
w
M, in H~POI-N~HzPOI
, 0.1
E .700
.600
.500
.400
.300
.200
.lOO
250
200 FIG.
buffer,
5. Absorption 0.1 M: O-O
spectra of Py5PINII, 0.5 X 1OW M, pH 5.4, l - - -0 pH 7.4.
i?sodium
so0
.400
.300
.200
.lOO
FIN. 6. Absorption spectra pH 5, l - - -0 pH 6.6.
of PySPCN, 305
10-4 M, in citrate
buffer,
0.1 M:
phosphat,e
E
.3ao
.200
-101
Il’rc. pH9.1,
0
7. Absorption spectra e- - -a pH 11.1.
of PySPCN,
1W M, in glycine-N&OH,
FIG. 8. Fluorm~~?nm npect~raof PySPCN, 0.4 X 10-r M, in metate -0 pH 5, l - - . l pH 6.2. Activation wavelength: 313 m#.
306
0,l .&f: o---o
buffer,
0.1 M:
320
350
400
410
9. Fluorescence spectra of Py5PCN, M : O--O pH 1, l - - -0 pH 3. Activation FIG.
TABLE pK:
Values of Puridozal -
Prim-
Pyridoxal 5-phosphate Pyridoxamine 5-phosphate Isonicotinoylhydrazone of pyridoxal 5-phosphate Cyanohydrin of pyridoxal 5-phosphate
and Derivatives
-
groups
Phenolic hydroxyl
gecondary phosphate
Pyridine nitro-
<2.50 (2.50 2.75
4.14 3.69 4.35
6.20 5.76 6.67
8.69 8.61 8.51
:2.50*
5.93
-
9.85
ary phosphate ____-
gen
1imino nitrogen
10.92 -
References
(7)a (7)a (This paper) (This
a The pK, values of Ref. (7) were determined at 25°C. in 0.15 M NaCl. b This value was determined by spectrofluorometric analysis. 307
mJ4
I
6-Phosphate Ionizable
Compounds
SW
0.4 X 10-r M, in HaPOd-NaHtPOa, wavelength: 315 mp.
paper)
308
BONAVITA AND SCARDI DISCUSSION
Table I shows the pK,’ values of pyridoxal 5-phosphate and pyridoxamine 5-phosphate, as measured by Williams and Neilands (7) and those of Py5PINH and Py5PCN, as determined in the present investigation. It is interesting to note that the pK,’ values for Py5PINH are not dissimilar from those corresponding to Py5P. The pK,’ of the primary and secondary phosphate groups, as well as of the phenolic hydroxyl group, are slightly higher than in Py5P, while the pK,’ of pyridine nitrogen is lower. Williams and Neilands (7) believe that the capacit,y of the 4-formyl group to withdraw electrons should facilitate the escape of protons from ortho and para positions. One should expect therefore that the disappearance of this formyl group should be reflected in a change of the pK,’ values corresponding to positions 3 and 1. There is, however, but a slight change when the formyl group is substituted by INH. The acidity of the phenolic hydroxyl decreasesin the order from pyridoxamine 5-phosphate to Py5P, Py5PINH, and Py5PCN. In the case of pyridoxamine 5-phosphat,e, the acidity is increased by the near posit,ively charged amino group. In the case of Py5PINH, the electron-withdrawing double-bond system in conjugation with the heterocyclic ring has been maintained as well as in Py5P, and the pK,’ value is very similar. The loss of the double-bond system in Py5PCN makes the phenolic hydroxyl less dissociable. A mathematical analysis for the pyridine nitrogen dissociation has given rt = 1, while in Py5PINH there are two pyridine nitrogen atoms, the former in the PySP molecule and the latter in isoniazid. The dissociation of the latter pyridine nitrogen may be supposed not to be spectrophotometrically evident at this pH, since even in INH it is not, evident. Williams and Neilands (7) have not observed spectral changes for Py5P and pyridoxamine 5-phosphate in relation to the dissociation of the primary and secondary phosphates. Py5PINH has not given a clear spectral evidence of these dissociations in the range 250420 rnp. An accurate analysis, however, has enabled useful spectral changes to be observed in the range 200280 rnp, from which the pK,’ values of the phosphate groups have been calculated. While primary and secondary phosphate groups are well insulated from the aromatic ring of PySP, they seem to be influenced by INH. It is interesting to note that the Py5PINH molecule, as a whole, seemsnot to be very different from Py5P, with respect to its ionizable groups. It seems likely that both configurations, the colored quinoid form and the alcoholic colorless form, which Hey1 et al. (8) have suggested for Py5P, are possible also for Py5PINH. The compound varies, between pH 1 and 11, from the colorless to a yellow form, even more colored than Py5P.
DERIVATIVES
OF PYRIDOXAL &PHOSPHATE
309
On the contrary, Py5PCN differs greatly from Py5P. It has not been possible to find spectral evidence of dissociation of the phosphate groups through an accurate analysis in the far ultraviolet. The pK,’ values both of the phenolic hydroxyl group and of the pyridine nitrogen appeared to be clearly higher than in Py5P. It seemscorrect to regard the pK,’ = 5.93 as due to the phenolic hydroxyl, since the dissociation of the secondary phosphate in PySP does not determine clear spectral changes. A similar consideration holds for pK,’ = 9.85, which we consider as due to the pyridine nitrogen and not to the CN- group. It must be pointed out that also the vitamin-Be derivatives studied by Williams and Neilands (7) showed a shift of the band around 325 rnp toward shorter wavelengths, in relation to the dissociation of the pyridine nitrogen. At variance with what has been observed with Py5PINH, the modification of the 4-position in Py5PCN caused a large increase in the pK,’ of both phenolic hydroxyl group and pyridine nitrogen. This points to quite a different form of compound. Investigations on the catalytic propert,ies of Py5P, Py5PINH, and Py5PCN substantiate this difference (3). ACKNOWLEDGMENTS The present investigation was made possible by grants of the Rockefeller Foundation and the Consiglio Nazionale delle Ricerche (Roma). We thank Dr. J. B. Neilands (Berkeley, California) for critical revision of the manuscript.
SUMMARY
1. The pK,’ values of the isonicotinoylhydrazone and the cyanohydrin of pyridoxal 5-phosphate were determined spectrophotometrically. 2. The apparent dissociation constants are very similar for pyridoxal 5-phosphate and its isonicotinoylhydrazone. 3. The cyanohydrin of pyridoxal 5-phosphate shows distinctly higher pK,’ values of the phenolic hydroxyl group and of the pyridine nitrogen. 4. An interpretation of these data has been attempted. REFERENCES 1. HONAVITA, 2. BONAVITA, 3. BOXAVITA, 4. SAH, P. P. 5. DAVISON, 6. BONAVITA, 7. WILLIAMS, 8. HEYL, D.,
(1951).
V., AND SCARVI, V., Eqerienlia 14.7 (1958). V., AND SCARDI, V., Experientia 14, 133 (1958). V., AND SCARDI, V., in press. T., J. Am. Chem. Sot. 76,300 (1954). A. N., Biochim. et Biophys. Acta 19, 131 (1956). V., AND SCARDI, V., Anal. Chim. Acta 20, 47 (1959). V. R., ANI) NEILANDS, J. B., Arch. Biochem. Biophys. 63, 56 (1954). LUR, E., HARRIS, S. A., ANI) FOLKERS, K., J. Am.. Chem. Sot. 73, 3430
OF
BIOCHEMISTRY
AND
BIOPHYSICS
300-309 (1959)
82,
Studies on Glutamic-Oxalacetic Transaminase. II. The Properties of Two Derivatives of Pyridoxal 5-Phosphate Vincenzo Bonavita and Vincenzo Scardi From
the Laboratory
of Human
Physiology,
Received
October
University
oj Ferrara,
Ferrara,
Italy
2, 1958
INTRODUCTION
A complete study on the properties of the isonicotinoylhydrazone and the cyanohydrin of pyridoxal Sphosphate (Py5P) has been undertaken in order to elucidate the mechanism of the interaction between Py5P and its apoenzymes (l-3). The present paper deals with results on the ionizable groups of both compounds, as compared with the native coenzyme, and on their spectrofluorometric properties. EXPERIMENTAL Pyridoxal 5-phosphate (Hoffmann-LaRoche) and isonieotinic acid hydrazide Lepetit (Nicotibina Lepetit, in crystals) have been used. The isonicotinoylhydrazone of Py5P (PyBPINH) has been prepared by direct reaction of stoichiometric amounts of both reactants, following the method suggested by Sah (4) for thechemical synthesis of the isonicotinoylhydraaone of pyridoxal hydrochloride. The yield was 6@64oj The reaction between Py5P and isoniazid (INH) in sodium phosphate buffer 0.05 M at different pH values has been followed as well in the experiments of Davison (5), in a Beckman spectrophotometer, model DU, equipped with a thermospacer. Figure 1 shows the rate of formation of Py5PINH with excess INH at 17” C., at pH values ranging from 5.0 s 8.0. It is evident that the rate is higher at the lower pH. The activation energy was found equal to 8700 cal./mole. This value does not agree with the value found by Davison (5) (14,000 cal./mole). The melting point is >230” C. And. Calcd. for C~~HISOBN~P: C, 46; H, 4.1; N, 15.3; P, 8.45. Found: C, 44.87; H, 4.23; N, 15.05; P, 7.95. The cyanohydrin of Py5P (Py5PCN) has been prepared by us as previously described (1, 6). The absorption spectra have been measured at room temperature (18” C.) in a Beckman spectrophotometer, model DU. Fluorescence spectra have been taken with a Farrand spectrofluorometer. Absorption and fluorescence spectra were measured at intervals of 0.4 pH unit, between 1 and 11, in order to obtain spectrophotometrically evident pK,’ values. pH measurements were made with a Beckman potentiometer. 300
DERIVATIVES
301
OF PYRIDOXAL &PHOSPHATE RESULTS
Absorption Spectra at Diferent
pH Values
Figure 2 shows the absorption spectra of Py5PINH at pH’s 3 and 5.2. The extinction maximum shifts from 300 rnp at pH 3 to 290 rnp at pH 5.2. The difference, Es40 - EzsO, was chosen for the calculation of this pK,‘. Figure 3 shows the absorption spectra of the same compound at pH’s 7 and 9. A shift of the extinction maximum from 292 to 310 rnp is evident. The absorption spectrum at pH 9 shows an almost complete fusion of the bands which at neutral pH have distinct maxima at 292 and at 340 mp. The difference, Em - Es30 , was chosen for the pK,’ calculation.
E292
.700
so0
.3oc
.lOC
5
10
FIG. 1. Rat,e of interaction between Py5P and INH at different 5 (0), pH 6 (O), pH 7 (o), pH 8 (X). INN excess: 25 times.
min.
pH values: pH
302
BONAVITA
AND
SCARD~
Figure 4 shows the absorption spectra of Py5PINH in the range 200280 mr, at pH’s 1 and 3. The difference, Ez6” - A&, , was chosen for this calculation. The pK,’ value was also confirmed from measurement of E 330 - E340 . In the former instance a pK,’ of 2.75, in the latter of 2.80, was found. Figure 5 shows the absorption spectra of Py5PINH between 200 and 280 rnp, at pH’s 5.4 and 7.4. The difference, Es,, - Ezo3, was chosen for the calculation. Figure 6 shows the absorption spectra of Py5PCN at pH’s 5 and 6.6. The difference, E2To- Ezoo, was chosen for the pK,’ calculation. Figure 7 shows the absorption spectra of the same compound at pH’a 9.1 and 11.1, with a shift of the extinction maximum from 318 to 308 mp. The difference, E320- E310, was chosen.
E 1.300
1.100
.900
.700
so0
,300
300 250 FIG. 2. Absorption spectra pH 3, .- - -0 pH 5.2.
300 of Py5PINH,
400
350 W4
M, in
acetate
m)r
buffer,
0.1
M: O--O
DERIVATIVES
OF
PYRIDOXAL
303
&PHOSPHATE
Fluorescence Spectra at Diflerent pH Values Fluorescence spectra of Py5P and Py5PINH have shown a great similarity. Both compounds, however, are not highly fluorescent in the pH range l-11. Py5PCN shows a fluorescence spectrum of peculiar interest, which has permitted the measurement of a pK,’ value around 5.85, very similar to that derived from the absorption specka (5.93). Figure 8 shows the fluorescence spectra of Py5PCN at pH’s 5 and 6.2. This quenching from neutralization has permitted us to define as highly fluorescent the form of the cyanohydrin having the phenolic hydroxyl group uncharged.
250 FIG.
pH7,
3. Absorption l --- l pH9.
300
spectra
350
of PyBPINH,
400
w
1W’ M, in tris-HCl,
0.1 M:
O--O
304
BONAVITA
AND
SCARDI
Figure 9 shows the fluorescence spectra of Py5PCN at pH’s 1 and 3. There is a clear quenching from excessive hydrogen-ion concentration, which has permitted us to measure a pK,’ < 2.50, evidently due to the dissociation of the primary phosphate group. This pK,’ is not evident from the absorption spectra. The fluorescence spectra at higher pH values have shown a gradual quenching but it was not possible to measure further pK,’ values. E .soo
.800
.600
. 500
.400
.300
.200
*loo
250
200
4. Absorption spectra M:O--OpHl, l ---•pH3. FIG.
of Py5PINH,
0.5 X 10-”
w
M, in H~POI-N~HzPOI
, 0.1
E .700
.600
.500
.400
.300
.200
.lOO
250
200 FIG.
buffer,
5. Absorption 0.1 M: O-O
spectra of Py5PINII, 0.5 X 1OW M, pH 5.4, l - - -0 pH 7.4.
i?sodium
so0
.400
.300
.200
.lOO
FIN. 6. Absorption spectra pH 5, l - - -0 pH 6.6.
of PySPCN, 305
10-4 M, in citrate
buffer,
0.1 M:
phosphat,e
E
.3ao
.200
-101
Il’rc. pH9.1,
0
7. Absorption spectra e- - -a pH 11.1.
of PySPCN,
1W M, in glycine-N&OH,
FIG. 8. Fluorm~~?nm npect~raof PySPCN, 0.4 X 10-r M, in metate -0 pH 5, l - - . l pH 6.2. Activation wavelength: 313 m#.
306
0,l .&f: o---o
buffer,
0.1 M:
320
350
400
410
9. Fluorescence spectra of Py5PCN, M : O--O pH 1, l - - -0 pH 3. Activation FIG.
TABLE pK:
Values of Puridozal -
Prim-
Pyridoxal 5-phosphate Pyridoxamine 5-phosphate Isonicotinoylhydrazone of pyridoxal 5-phosphate Cyanohydrin of pyridoxal 5-phosphate
and Derivatives
-
groups
Phenolic hydroxyl
gecondary phosphate
Pyridine nitro-
<2.50 (2.50 2.75
4.14 3.69 4.35
6.20 5.76 6.67
8.69 8.61 8.51
:2.50*
5.93
-
9.85
ary phosphate ____-
gen
1imino nitrogen
10.92 -
References
(7)a (7)a (This paper) (This
a The pK, values of Ref. (7) were determined at 25°C. in 0.15 M NaCl. b This value was determined by spectrofluorometric analysis. 307
mJ4
I
6-Phosphate Ionizable
Compounds
SW
0.4 X 10-r M, in HaPOd-NaHtPOa, wavelength: 315 mp.
paper)
308
BONAVITA AND SCARDI DISCUSSION
Table I shows the pK,’ values of pyridoxal 5-phosphate and pyridoxamine 5-phosphate, as measured by Williams and Neilands (7) and those of Py5PINH and Py5PCN, as determined in the present investigation. It is interesting to note that the pK,’ values for Py5PINH are not dissimilar from those corresponding to Py5P. The pK,’ of the primary and secondary phosphate groups, as well as of the phenolic hydroxyl group, are slightly higher than in Py5P, while the pK,’ of pyridine nitrogen is lower. Williams and Neilands (7) believe that the capacit,y of the 4-formyl group to withdraw electrons should facilitate the escape of protons from ortho and para positions. One should expect therefore that the disappearance of this formyl group should be reflected in a change of the pK,’ values corresponding to positions 3 and 1. There is, however, but a slight change when the formyl group is substituted by INH. The acidity of the phenolic hydroxyl decreasesin the order from pyridoxamine 5-phosphate to Py5P, Py5PINH, and Py5PCN. In the case of pyridoxamine 5-phosphat,e, the acidity is increased by the near posit,ively charged amino group. In the case of Py5PINH, the electron-withdrawing double-bond system in conjugation with the heterocyclic ring has been maintained as well as in Py5P, and the pK,’ value is very similar. The loss of the double-bond system in Py5PCN makes the phenolic hydroxyl less dissociable. A mathematical analysis for the pyridine nitrogen dissociation has given rt = 1, while in Py5PINH there are two pyridine nitrogen atoms, the former in the PySP molecule and the latter in isoniazid. The dissociation of the latter pyridine nitrogen may be supposed not to be spectrophotometrically evident at this pH, since even in INH it is not, evident. Williams and Neilands (7) have not observed spectral changes for Py5P and pyridoxamine 5-phosphate in relation to the dissociation of the primary and secondary phosphates. Py5PINH has not given a clear spectral evidence of these dissociations in the range 250420 rnp. An accurate analysis, however, has enabled useful spectral changes to be observed in the range 200280 rnp, from which the pK,’ values of the phosphate groups have been calculated. While primary and secondary phosphate groups are well insulated from the aromatic ring of PySP, they seem to be influenced by INH. It is interesting to note that the Py5PINH molecule, as a whole, seemsnot to be very different from Py5P, with respect to its ionizable groups. It seems likely that both configurations, the colored quinoid form and the alcoholic colorless form, which Hey1 et al. (8) have suggested for Py5P, are possible also for Py5PINH. The compound varies, between pH 1 and 11, from the colorless to a yellow form, even more colored than Py5P.
DERIVATIVES
OF PYRIDOXAL &PHOSPHATE
309
On the contrary, Py5PCN differs greatly from Py5P. It has not been possible to find spectral evidence of dissociation of the phosphate groups through an accurate analysis in the far ultraviolet. The pK,’ values both of the phenolic hydroxyl group and of the pyridine nitrogen appeared to be clearly higher than in Py5P. It seemscorrect to regard the pK,’ = 5.93 as due to the phenolic hydroxyl, since the dissociation of the secondary phosphate in PySP does not determine clear spectral changes. A similar consideration holds for pK,’ = 9.85, which we consider as due to the pyridine nitrogen and not to the CN- group. It must be pointed out that also the vitamin-Be derivatives studied by Williams and Neilands (7) showed a shift of the band around 325 rnp toward shorter wavelengths, in relation to the dissociation of the pyridine nitrogen. At variance with what has been observed with Py5PINH, the modification of the 4-position in Py5PCN caused a large increase in the pK,’ of both phenolic hydroxyl group and pyridine nitrogen. This points to quite a different form of compound. Investigations on the catalytic propert,ies of Py5P, Py5PINH, and Py5PCN substantiate this difference (3). ACKNOWLEDGMENTS The present investigation was made possible by grants of the Rockefeller Foundation and the Consiglio Nazionale delle Ricerche (Roma). We thank Dr. J. B. Neilands (Berkeley, California) for critical revision of the manuscript.
SUMMARY
1. The pK,’ values of the isonicotinoylhydrazone and the cyanohydrin of pyridoxal 5-phosphate were determined spectrophotometrically. 2. The apparent dissociation constants are very similar for pyridoxal 5-phosphate and its isonicotinoylhydrazone. 3. The cyanohydrin of pyridoxal 5-phosphate shows distinctly higher pK,’ values of the phenolic hydroxyl group and of the pyridine nitrogen. 4. An interpretation of these data has been attempted. REFERENCES 1. HONAVITA, 2. BONAVITA, 3. BOXAVITA, 4. SAH, P. P. 5. DAVISON, 6. BONAVITA, 7. WILLIAMS, 8. HEYL, D.,
(1951).
V., AND SCARVI, V., Eqerienlia 14.7 (1958). V., AND SCARDI, V., Experientia 14, 133 (1958). V., AND SCARDI, V., in press. T., J. Am. Chem. Sot. 76,300 (1954). A. N., Biochim. et Biophys. Acta 19, 131 (1956). V., AND SCARDI, V., Anal. Chim. Acta 20, 47 (1959). V. R., ANI) NEILANDS, J. B., Arch. Biochem. Biophys. 63, 56 (1954). LUR, E., HARRIS, S. A., ANI) FOLKERS, K., J. Am.. Chem. Sot. 73, 3430