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The reaction between cysteamine and pyridoxal phosphate
Letters The Reaction
between
Pyridoxal
Cysteamine
to the
Editors
was 10m3N and that of pyridoxal phosphate, 10-I M. All the solutions were prepared in water distilled in an all-glass apparatus. At zero time, and at, various incubation times, spectrophotometric analyses of the incubation mixture were performed in a Beckman instrument. The results obtained are reported in Figs. 1 and 2. Cysteamine readily links to pyridoxal phosphate giving rise t,o a thiazolidine derivative as demonstrated by a change of the spectrophotometric curve of ppridoxal phosphate. The maximum at 390 mp disappeared, and a peak at 330 rnp, chara+ teristic of the thiazolidinc ring (2, 3), became evident. -%fter incubation, the spectrophotometric curve is again changed. The peak at 330 rn+ disappeared and a new peak appeared with a maximum at 410 mp, specific for the Schiff bases of pyridoxal with amino acids. The reaction is more rapid at pH 8. The oxidat,ion of cysteaminc. as evident by the disnppear-
and
Phosphate
Recently we observed that cysteamine causes a transient inhibition of the oxidation of cadaverine, histamine, and cystamine by diamine oxidase (1). The inhibition showed a characteristic feature since it disappeared after about 1 hr. of incubation, at which time cysteamine was spontaneously and completely oxidized to cystamine. In order to explain these results, it could be supposed that cysteaminc inhibition is due to the coupling of the thiol with pyridoxal phosphate in a thiazolidinc ring. Oxidation of cysteamine is nevertheless not prevented, and when all thr thiol is transformed into the corresponding disulfide, pyridoxal phosphate is again free for enzymic activit,y. To test this hypothesis an excess of cystcaminc was incubated at 38°C. with pyridoxal phosphate in 0.1 M phosphate buffers at pH values of 6.8, 7.4, and 8.0. The final concentration of cysteamine
1.c)l
IO Q.D.
B
0. D.
06.
330 ‘/
250
300
390
350
1
400
L
r
450 mp
250
300
350
400
450 mp
Fro. 1. Broken lines, lo-’ M pyridoxal phosphate in 0.1 M phosphate buffer pH 8. Solid lines, lo-” di l)yridoxal phosphate and 10.” dl cystrarninc in 0.1 .I1 phosphate buffer pH 8; 9: 5 min. after mixing: b. After 30 min. of incubation nt 38°C. in a water bath with agitation. 526
LETTERS
L-__,
1
TO THl? EDITORS
L-
~-
2 hrs
~-
~.-
---...
2 hrs
1
FIG. 2. Variation with time of the optical density at 330 rnp (A) and 410 mk (R) of solutions containing lo-’ &I pyridoxal phosphate and 1O‘3&I cysteamine, at the indicated pH. The solutions were incubated directly in the Beckman syectrophotomcter thcrmorcgulntcd at 38°C. ante of the peak at 330 mfi or by the increase in O.D. at 410 rnp, is nearly complete in 30 min. at ~IH 8 or 7.4, but requires about 2 hr. at pH 6.8. The complete oxidation of cysteamine was also checked in a Warburg apparatus. The oxygen consumption reached the theoretical value of 0.25 mole &/mole SW, with time curves at the three pH values studied, paralleling the changes in O.D. at 330 or 410 w. It must be concluded that cysteamine interacts reversibly with pyridoxal phosphate to form a dissociable thiazolidine derivative in equilibrium with the unreacted cysteamine, which is in turn spontaneously oxidized to cystamine. When all the thiol is transformed to the corresponding disulfide, the thiazolidine complex of pyridoxal disappears and the latter links to cystamine to form a Schiffbase:.In the light of the present data, the transient inhibition that cysteamine could manifest versus pyridoxal phosphate-dependent enzymes, such as diamine oxidxse, is understandable. Inhibition is observed as long as pyridoxal phosphate is bound in the form of a thiazolidine ring, and is reversed when oxidation of the thiol liberates the pyridoxal. ACKNOWLEDGMEKIS The Aut,hors are grateful to Miss M. T. Graziani for skilled technical assistance.
1.
DE
h’f.4~~0,
C.,
Mosnovi,
B., AED C~VA~LISI,
D.,
Bioch,em. Pha~macol. in press (1962). 2. bhTSU0, I’., J. Am. Chcm. ,%c. 79, 2011 (1957).
3. BUELL,
M. V.,
sot. 82,6042
ASD
HAKSRN,R. E., J.
Am.
Chem.
(1960).
C. De D.
MARCO Bocro~o
lstituto di Chimica Uiologica Uniuersit& di Roma, Italy Rcceiued dla?~ 23, IX??
Polarographic
Behavior
of
Cytosine
In 1946 Heath (1) found that of all nucleic acid bases only adenine and/or its nucleosides and nurleot,ides produce a polarographic reduction awe. Some of Heath’s findings have been confirmed by Cavalirri et al. (2) and by Gh;tz-Luthy et al. (3). Later, one of us (E.P.) was engaged in studying the oscillopolarographic behavior of nucleic acid components when polarizing with alternating Furrent, and found that all bases yield indentations on the dE/dt = f(E) curves (4). The oscillographic behavior of some bases could be explained on the basis of electrode processes, such as artifacts and capacity processes (4, 5) specific to alternatingcurrent oscillographic polarography. The behavior of c>%osine and its nucleosides did not, homcrer, corwspond to the criteria rharacteristic of elcct,rodc processes of this kind, but it rather gave w-itlcnce in favor of some kind of reduction process ~wually oacuring in classic pol:arography. where the mercury drop electrode i,* polarized by direct voltage. For this re:won, wc dwidrcl to \-crif,v Heath’s findings (1).
between
Pyridoxal
Cysteamine
to the
Editors
was 10m3N and that of pyridoxal phosphate, 10-I M. All the solutions were prepared in water distilled in an all-glass apparatus. At zero time, and at, various incubation times, spectrophotometric analyses of the incubation mixture were performed in a Beckman instrument. The results obtained are reported in Figs. 1 and 2. Cysteamine readily links to pyridoxal phosphate giving rise t,o a thiazolidine derivative as demonstrated by a change of the spectrophotometric curve of ppridoxal phosphate. The maximum at 390 mp disappeared, and a peak at 330 rnp, chara+ teristic of the thiazolidinc ring (2, 3), became evident. -%fter incubation, the spectrophotometric curve is again changed. The peak at 330 rn+ disappeared and a new peak appeared with a maximum at 410 mp, specific for the Schiff bases of pyridoxal with amino acids. The reaction is more rapid at pH 8. The oxidat,ion of cysteaminc. as evident by the disnppear-
and
Phosphate
Recently we observed that cysteamine causes a transient inhibition of the oxidation of cadaverine, histamine, and cystamine by diamine oxidase (1). The inhibition showed a characteristic feature since it disappeared after about 1 hr. of incubation, at which time cysteamine was spontaneously and completely oxidized to cystamine. In order to explain these results, it could be supposed that cysteaminc inhibition is due to the coupling of the thiol with pyridoxal phosphate in a thiazolidinc ring. Oxidation of cysteamine is nevertheless not prevented, and when all thr thiol is transformed into the corresponding disulfide, pyridoxal phosphate is again free for enzymic activit,y. To test this hypothesis an excess of cystcaminc was incubated at 38°C. with pyridoxal phosphate in 0.1 M phosphate buffers at pH values of 6.8, 7.4, and 8.0. The final concentration of cysteamine
1.c)l
IO Q.D.
B
0. D.
06.
330 ‘/
250
300
390
350
1
400
L
r
450 mp
250
300
350
400
450 mp
Fro. 1. Broken lines, lo-’ M pyridoxal phosphate in 0.1 M phosphate buffer pH 8. Solid lines, lo-” di l)yridoxal phosphate and 10.” dl cystrarninc in 0.1 .I1 phosphate buffer pH 8; 9: 5 min. after mixing: b. After 30 min. of incubation nt 38°C. in a water bath with agitation. 526
LETTERS
L-__,
1
TO THl? EDITORS
L-
~-
2 hrs
~-
~.-
---...
2 hrs
1
FIG. 2. Variation with time of the optical density at 330 rnp (A) and 410 mk (R) of solutions containing lo-’ &I pyridoxal phosphate and 1O‘3&I cysteamine, at the indicated pH. The solutions were incubated directly in the Beckman syectrophotomcter thcrmorcgulntcd at 38°C. ante of the peak at 330 mfi or by the increase in O.D. at 410 rnp, is nearly complete in 30 min. at ~IH 8 or 7.4, but requires about 2 hr. at pH 6.8. The complete oxidation of cysteamine was also checked in a Warburg apparatus. The oxygen consumption reached the theoretical value of 0.25 mole &/mole SW, with time curves at the three pH values studied, paralleling the changes in O.D. at 330 or 410 w. It must be concluded that cysteamine interacts reversibly with pyridoxal phosphate to form a dissociable thiazolidine derivative in equilibrium with the unreacted cysteamine, which is in turn spontaneously oxidized to cystamine. When all the thiol is transformed to the corresponding disulfide, the thiazolidine complex of pyridoxal disappears and the latter links to cystamine to form a Schiffbase:.In the light of the present data, the transient inhibition that cysteamine could manifest versus pyridoxal phosphate-dependent enzymes, such as diamine oxidxse, is understandable. Inhibition is observed as long as pyridoxal phosphate is bound in the form of a thiazolidine ring, and is reversed when oxidation of the thiol liberates the pyridoxal. ACKNOWLEDGMEKIS The Aut,hors are grateful to Miss M. T. Graziani for skilled technical assistance.
1.
DE
h’f.4~~0,
C.,
Mosnovi,
B., AED C~VA~LISI,
D.,
Bioch,em. Pha~macol. in press (1962). 2. bhTSU0, I’., J. Am. Chcm. ,%c. 79, 2011 (1957).
3. BUELL,
M. V.,
sot. 82,6042
ASD
HAKSRN,R. E., J.
Am.
Chem.
(1960).
C. De D.
MARCO Bocro~o
lstituto di Chimica Uiologica Uniuersit& di Roma, Italy Rcceiued dla?~ 23, IX??
Polarographic
Behavior
of
Cytosine
In 1946 Heath (1) found that of all nucleic acid bases only adenine and/or its nucleosides and nurleot,ides produce a polarographic reduction awe. Some of Heath’s findings have been confirmed by Cavalirri et al. (2) and by Gh;tz-Luthy et al. (3). Later, one of us (E.P.) was engaged in studying the oscillopolarographic behavior of nucleic acid components when polarizing with alternating Furrent, and found that all bases yield indentations on the dE/dt = f(E) curves (4). The oscillographic behavior of some bases could be explained on the basis of electrode processes, such as artifacts and capacity processes (4, 5) specific to alternatingcurrent oscillographic polarography. The behavior of c>%osine and its nucleosides did not, homcrer, corwspond to the criteria rharacteristic of elcct,rodc processes of this kind, but it rather gave w-itlcnce in favor of some kind of reduction process ~wually oacuring in classic pol:arography. where the mercury drop electrode i,* polarized by direct voltage. For this re:won, wc dwidrcl to \-crif,v Heath’s findings (1).