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Polarographic behavior of cytosine
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).
SfL8
LETTERS
TO THE
Icor our experiments we usrd He~~vsk~‘s polarograph, type LP 55, and a (hopping inetwry elec1rod<, in Iinlousek’s cell wvltll a Sepitr~ltCd normal c*nlomcl electrode. While gunnine, thymine, and IlraAl, in agreement with Hcnth’s findings (l), did not, product any polarogrnpl~~c rcduclion, cytosine, (sy1iclinc,, df~osycytidine. ant1 cytidylic and deoxycytidylic. acids yielded :I reduction WUYV with E1j3 al atlout -1.6 V. at neutr:d pH, the wax-e’s height being linearly dependent on concentration, and within a rathrlr wide mngc, independent of pH (Fig. 1). In a highly acid mrdium, t,he ware fuses with the curve of h,vdrogen elimination. Conscc~uontly, i,his might, hare cxasily rs~ped Heath’s attention, because hi:: polarogl,:lphic analysis was performc,cI in a medium of 0.1 S HClO., (1). The height of the waves is practic~ally independent of buffrxr concmtraf ion and r:tric.q lincnl,ly with the sctun~ root of the mrrcur>. c.olumn hr~ight, which, iclr~nticnlly with thr pr(~cf~ding d:lt:l. suggests that 1Il(s limiting currf’nt is rt~guhtlctl by tliffusion. A similar polsrogmphic behavior was also observed in the case of 5-methylcytosine and 5-hytlros~methylcytosinc. Experiments nimctl at prclpnring and iclcntifying a reduction pl’oduct ha\-e not, yet been c~ompl<~tc~l. It appears, howrr~cr, that the double bond --C=Sin position 1,6 is rctluccd, which wcndtl :tgrec ~~11 \vith the, f:u,t that c,ytosinc, bound in nati\,fi tfr,os~ril-)onut,lei(. ac,icl (DNi\ ) is not r+ tlu~tl I)ol:lrogl.nI)l1i~:lll.‘. c6), since, this clollbl(, I)ontl
I’IG. 1. Dependence of the cgtidine wave on pH. Cytidine, 2 X 10m4M. Hritton-Robinson buffers of pH: 1, 2.6; 2, 3.0; 3, 3.6 from -1.1 v.; 4, 4.1; 5, 4.6; 6, 4.9 from -1.2 v.; 7, 5.6; 8, 6.0 from -1.3 v.; 9, 6.6; 10, 7.0 from -1.4 v.; 11, 7.7; 12, 8.0 from -1.5 v.; 13, 8.5; 14, 9.0 from -1.6 v.; 15, 10.0 from -1.7 v.; 16, 10.8 from -1.8 v.; N.C.E., h = 42 cm., sens. l/30, 100 mv./abscissa.
EDITORS
is folmtl \vitllin IVlf~.
6. PAL~~&K,
the tlouble
IX.. Hiochitn.
helix of the DNA
n~ole-
et 1Gophy.s. Actn
51, 1
(1961).
Some Properties
of
Human
Trypsin’
In previous publications from this laboratory (1, 2) it has been rrported that porcine trypsin differs markedly in cc,rtain respects from the trypsins isolated from the pancreas of two ruminants, ox and sheep. To characterize trypsin from some 01her nonruminant species, for comparison with thcx cnzymes previously studied, the purification of human trypsin has been undertakm. The proper1 its of the resultant partially purified enzyme will br: relmrtptl here,. amounts of human pnncrea.G Since only s~nall wr’re available, crystallization of the enzyme by the standard method was impractical. The panc’reas was mincetl. extracted with cold 0.25 11 ,qulfuril. acid, and tllc, f,xtract was fractionatc,d wvlth ammonium sulfate in the conventional mnnn~r (3). The fraction obtained bcstwrrn 0.4 and 0.7 rat\lrat ion (about 2 g. from 400 g. of p:rnclcns) writs t hc’rl worktd up for tr>‘lAn by thr following sclrr~mc’ : (1) One g. of the F)rc.c*ipitnte was d&sol\-catI in 50 -~ ~~ -____-_1 Communication So. 385. This study was supportend in part tly grants from t,hp Sationnl Scien~ Fountlntion and the, I-. S. Atomic Enrlrgy Commission. Tlln,nke arc’ due to Mr. 8. .J. Vithaynthil for carrying out tllcs electrophorcsis rims. The: sarnplc,r of human panc’rc’as were obtained through the courlrsy of Dr. Angelo Gnassi, Pathologist-inChief, Medical Center, Jersey Cil,y, New Jersey. The Sephades used was a gift of Pharmaria Fine: Chemicals, Inc., Rochester, Minn.
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).
SfL8
LETTERS
TO THE
Icor our experiments we usrd He~~vsk~‘s polarograph, type LP 55, and a (hopping inetwry elec1rod<, in Iinlousek’s cell wvltll a Sepitr~ltCd normal c*nlomcl electrode. While gunnine, thymine, and IlraAl, in agreement with Hcnth’s findings (l), did not, product any polarogrnpl~~c rcduclion, cytosine, (sy1iclinc,, df~osycytidine. ant1 cytidylic and deoxycytidylic. acids yielded :I reduction WUYV with E1j3 al atlout -1.6 V. at neutr:d pH, the wax-e’s height being linearly dependent on concentration, and within a rathrlr wide mngc, independent of pH (Fig. 1). In a highly acid mrdium, t,he ware fuses with the curve of h,vdrogen elimination. Conscc~uontly, i,his might, hare cxasily rs~ped Heath’s attention, because hi:: polarogl,:lphic analysis was performc,cI in a medium of 0.1 S HClO., (1). The height of the waves is practic~ally independent of buffrxr concmtraf ion and r:tric.q lincnl,ly with the sctun~ root of the mrrcur>. c.olumn hr~ight, which, iclr~nticnlly with thr pr(~cf~ding d:lt:l. suggests that 1Il(s limiting currf’nt is rt~guhtlctl by tliffusion. A similar polsrogmphic behavior was also observed in the case of 5-methylcytosine and 5-hytlros~methylcytosinc. Experiments nimctl at prclpnring and iclcntifying a reduction pl’oduct ha\-e not, yet been c~ompl<~tc~l. It appears, howrr~cr, that the double bond --C=Sin position 1,6 is rctluccd, which wcndtl :tgrec ~~11 \vith the, f:u,t that c,ytosinc, bound in nati\,fi tfr,os~ril-)onut,lei(. ac,icl (DNi\ ) is not r+ tlu~tl I)ol:lrogl.nI)l1i~:lll.‘. c6), since, this clollbl(, I)ontl
I’IG. 1. Dependence of the cgtidine wave on pH. Cytidine, 2 X 10m4M. Hritton-Robinson buffers of pH: 1, 2.6; 2, 3.0; 3, 3.6 from -1.1 v.; 4, 4.1; 5, 4.6; 6, 4.9 from -1.2 v.; 7, 5.6; 8, 6.0 from -1.3 v.; 9, 6.6; 10, 7.0 from -1.4 v.; 11, 7.7; 12, 8.0 from -1.5 v.; 13, 8.5; 14, 9.0 from -1.6 v.; 15, 10.0 from -1.7 v.; 16, 10.8 from -1.8 v.; N.C.E., h = 42 cm., sens. l/30, 100 mv./abscissa.
EDITORS
is folmtl \vitllin IVlf~.
6. PAL~~&K,
the tlouble
IX.. Hiochitn.
helix of the DNA
n~ole-
et 1Gophy.s. Actn
51, 1
(1961).
Some Properties
of
Human
Trypsin’
In previous publications from this laboratory (1, 2) it has been rrported that porcine trypsin differs markedly in cc,rtain respects from the trypsins isolated from the pancreas of two ruminants, ox and sheep. To characterize trypsin from some 01her nonruminant species, for comparison with thcx cnzymes previously studied, the purification of human trypsin has been undertakm. The proper1 its of the resultant partially purified enzyme will br: relmrtptl here,. amounts of human pnncrea.G Since only s~nall wr’re available, crystallization of the enzyme by the standard method was impractical. The panc’reas was mincetl. extracted with cold 0.25 11 ,qulfuril. acid, and tllc, f,xtract was fractionatc,d wvlth ammonium sulfate in the conventional mnnn~r (3). The fraction obtained bcstwrrn 0.4 and 0.7 rat\lrat ion (about 2 g. from 400 g. of p:rnclcns) writs t hc’rl worktd up for tr>‘lAn by thr following sclrr~mc’ : (1) One g. of the F)rc.c*ipitnte was d&sol\-catI in 50 -~ ~~ -____-_1 Communication So. 385. This study was supportend in part tly grants from t,hp Sationnl Scien~ Fountlntion and the, I-. S. Atomic Enrlrgy Commission. Tlln,nke arc’ due to Mr. 8. .J. Vithaynthil for carrying out tllcs electrophorcsis rims. The: sarnplc,r of human panc’rc’as were obtained through the courlrsy of Dr. Angelo Gnassi, Pathologist-inChief, Medical Center, Jersey Cil,y, New Jersey. The Sephades used was a gift of Pharmaria Fine: Chemicals, Inc., Rochester, Minn.