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The effect of radioprotective agents on tissue nonprotein sulfhydryl and disulfide levels
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Letters The Effect of Rodioprotective Nonprotein Sulfhydryl and
Agents Disulfide
98
(1962)
to the Editors
on Tissue Levels’
The radioprotective action of aminoethylthiosulfuric acid and aminoethplthiophosphate has been previously reported (1, 2), and the possibilit)y was considered that t,he protective action of these compounds was due to an in 2liz1o conversion of the thioesters to the corresponding sulfhydryl compound, cysteamine. The latter is a well-known mdioprotective compound. In order to test this hypot,hcsis the thiocsters now have been injected into mice and the nonprotein sulfhydryl and disulfide content of spleen, liver and blood has been dekrmined. Splern was chosen as an example of a radiosensitive tissue, liver represented a more radioresistant although metabolically very active tissue and blood was included for reasons drscribed later on. The in\-estigation was extended i o inrludc certain other protective and nonprotective compounds of interest in this conn&ion. The preparations of aminoethylthiosulfuric acid and nminoethylthiophosphate have been described previously (1. 2). Cystamine dihydrochloride was prepared from cystc‘nmine by oxidation with hpdrogm peroxide and recrystallizations from cthanol-hydrochloric arid. Other compounds were commercial products. The animals were female CBA-micr of 20 g. body weight. The compounds at dose levels reported (I-3) to give prot,ect,ion or, in case of thioglpc.olat,e and mrrcaptoethanol, no protection, WPI’P dissolved in saline and given hy intrappritoneal injection in a volume of 0.2 ml. J$%en lirrr and spleen were assayed, the animals were killed 10 min. after injection by immersion in an ethanolsolid rarbon dioxide mixture. Blood was analyzed in separate experiments. The samples in this case wcle obtained by heart puncturr also 10 min. after injection of the compound studied. Pooled tissue samples from 2-4 mice wpre then homogenized in ice-cooled 4% (w/l-) sulfosalirylic acid (lo-20 volumes per weight of tissue) in a glass Potter-Elvehjrm homogenizer. The homogenate was rentrifugcd and the sulfhydryl content of the, supernat,ant drtermined by a nitroprussidc mrlthod (4). In rasc of q&amine, aminoethylthiosulfuric arid, aminoethylthiophosphate, and nontreatcd controls deter‘This work has been supported by a grant from the Swedish Medical Rcsearc>h C’olmcil.
minations were also made by the dithiobisnitrohenzoic acid method (5), which had the advantage of giving stable absorbancy readings in contrast to the nitroprusside method. Satisfactory agreement (within 25%) was obtained always with the two methods. gttempts also were made to dctermine the sulfhydryl content by amperometric silver titration (6), but although glutathionc gave a sharp and reproducible end point, cystcamine did not, and Lhis method consequently was abandoned. Nonprotein disulfide compounds were determined on an aliquot of the sulfosalicylic acid extract tq electrolytic reduction (7) of the disulfitle to the corresponding sulfhydryl compound and determination of tho latter with nitroprusside. The thioesters were, howevrr, found to interferc7 in this determination as they also were reduced c>lcrtrolytically to the rorrrsponding sulfhydryl rompound. It was found (Table I) that injection of all the sulfur-containing compounds gave increased nonprotein sulfhydryl levels in the tissues studied. The differences were statistically significant (p < 0.05). It is rvident, from Table I that as the increases in liver and spleen were larger than thosr in blood, the increasrd values in liver and spleen could not be attrihutrd to the blood content of these tissues. It also was found that, the pharmacological a&-e amine serotonin (5-hydroxytrypktmine) which does not contain “potential” sulfhpdrpl groups, gave a slight, hut significant inrrease of sulfhydryl l~lvrl in spleen and liver (Table I). The effect of the compounds studied on t.hr nonprotein disulfide compounds of spleen and liver is shown in Table II. In this case no or J-cry small increases were ohservcd. Only in the liver after cystamine, mrrcaptoethanol, and aminoethylthiosulfuric acid and in the spleen nftrr t,hc, lust-mcntioned compound were significant incrclascs notr,tl (p < 0.05). The increased values observed after injection of the thioestcr may, howcvclr, hr due t,o the pre$mce of the parent compound itself, whirh intcrfrres with the disulfidc detrrmination, ii.5 *jrr>viously mentioned. Th(> results obtained in the presrnt investigation are of some interest in connection with the mrchanism of action of the radioprotcctive drugs. It has been suggested for instance that the radioprotcctive sulfhpdryl compounds owe their effert, to thrir ntA1it.v to react with radiation-induced free radicsalp
LETTERS
TO THE TABLE
Compound injected
fpmoles/g.)
+
0.14
(8)
200 200 360
6.31 + 0.18 6.3“ f 0.12 5.24 f 0.2i
(5) (5) (6)
400
5.89 i
(6)
265 150 50
5.69 f 0.073 (4) 6.38 f 0.13 (4) 3.88 IIZ 0.0% (6)
u Values given as mean f
standard
error.
0.20
Figures TBBLE
DISI-LFIDE
Compound injected
Cysteamine HCI Cystamine. 2HCl Aminoethylthiosulfuric acid Sodium aminoethylthiophosphate Sodium thioglycolate Mercaptoethanol Serotonin-creatinine sulfate a Values
Liver
Spleen 3.40
~ONI'ROTEIN
I
Sulfhydryl content
DOW bllg./kg.)
Cystenmine. HCI Cystamine. 2HCl Arnirloethvlthioslllfuric acid Sodium aminoetjhythiophosphat,e Podium thioglycolate Mercaptoethanol Serotonincreatinine sulfat,e
given as mean & standard
343
EDITORS
r,EVELS
7.56
i
Blood
0.25
(8)
0.56
0.18 0.49 0.09
(5) (5) (6)
1.19 i 0.i-f
9.65 zk 0.23
(6)
1 .21 + 0.061
(4)
1.45 f
(4)
10.3 f 9.5i f X.88 f
8.29 It 0.18 (1) 10.2 It 0.15 (4) 8.29 zk 0.22 (6)
in parentheses
indicate
number
f
0.04
(4)
0.098
(1)
zt 0.049
0.043
of determinat~ions.
II
IN Tmsc-ES
OF TREATEI)
.4x1) COSTROL
~~I(W
DOS+? (mg.jkg.j
0.17 0.43 0.33 0.73 0.26 0.06 0.15 -0.01
200 200 360 400 265 150 5!) error.
Figures
(8). The present investigation showed that both the protective cysteamine and the nonprotective thioglpcolate and mercaptoethanol gave similar increases of the sulfhydryl concentration in the spleen. However, protective and nonprotective sulfhydryl compounds seem to be equally effective for inactivation of free radicals (3) and (intracellular distribution phenomena being disregarded) the present results thus do not support the “radicalscavenger” hypothesis. Other theories, such as the “mixed-disulfide” hypothesis (9) which take into account the difference in chemical reactivities of the different sulfhydryl compounds seem to be in better agreement with the results of the present investigation. Concerning the mechanism of action of the thioesters studied (aminoethylthiosulfuric acid and aminoethylthiophosphate) the present results suggest, that these compounds are converted in viva
f 0.063 f 0.15 f 0.11 z!c 0.086 2~ 0.13 f 0.033 Zt 0.13 + 0.039
in parentheses
(8) (5) (5) (6) (6) (4) (1) (6)
indicate
0.03 0.09 0.38 0.70 0.06 0.02 0.32 -0.01 number
f f + f + f + i
0 023 (8) 0.025 (5) 0.11 (5) 0.080 (6) 0.016 (6) 0.011 (4) 0.033 (4) 0.017 (6)
of tlrterminations.
to cysteamine, lvhich then may be the effective protective agent. These conversions are probably enaymic, and an enzymic hydrolysis of the thiophosphate has in fact been demonstrat,ed by Akerfeldt (10). In case of the thiosulfate ester, however, previous in zrilro studies (11) gave no evidence for an enzymic hydrolysis, and further experiments (unpublished) showed that if the compound was incubated with a mouse liver homogenate, no increase, but on the contrary a decrease of the nonprotein sulfhydryl concentration in the homogenate was observed. This effect is explained by the fact that, the thiosulfat)e ester reacts spontaneously with glutathione (11) to give the mixed disulfide of cpsteamine and glutathione. The latter may, however, be expected to be reduced to the free thiols through the action of glutathione reductase according to the mechanism elucidated by Pihl et al. (12). The existence of such a reac-
LETTERS
341
TO
tion was supported by the fact that, when a systcm containing liver homogenate and thiosulfate ester was supplemented with TPS plus glucose-6phosphate (which gives TPKH due t,o the presence of glucose-5-phosphntc dchydrogenase in the homogenate), an increase of nonprotein sulfhydryl groups was obscr\.rd. It is of interest that all the sulfur-containing protective compounds studied in this investigation, being either thiols, disulfides, or thioesters, gave increased nonprot,ein sulfhydryl levels in the tissues but only slight or no increases in disulfide levels. The rapid reduction of cystamine in viva to cysteamine has been noted previously by others (13, 14). If the radioprotective action of the sulfur-containing agents is due to a formation of mixed disulfides with tissue constit,uents, as suggcsted by Eldjarn and Pihl (91, the present results indicate that the mixed disulfides are formed by interaction of radiosmsitive disulfidc bonds in thr tissue and the protecti\-c agent. The latter would he a sulfhydryl compound from the beginning or converted to such a cornl)ound in the body. The sensitivity of disulfide bond,? i o ionizing radiation has in fact been notcad (3, 15). Finally, the fact that scrotonin also caused a slight increase in the sulfhydryl content of the spleen merits some discussion. The excellent work of van der Meer and van Bekkum (16) has demonstrated that the radioprotective effect of serotonin can be attribut,ed to s diminished “oxygen cart,” as serotonin caus(xs a pronoun& tissue hypoxia in the spleen and bone marrow. It is possible, however. that the incrrasc in nonprotein sulfhydryl compounds obsclr\-ed in t,he present investigation contributc>s to the r:tdioprotecti\-e rffeet of serotonin. REFERENCES 1. HOLMBERG, B., ASD S~RBO, B., Nature 183, 832 (1959). 2. H.~NSEN, B., ASD SGRBO, B., Acta Radial. 56, 141 (1961). 3. ELDJARK. L., .~ND PIHI,. A., In “Mechanisms in Radiobiology” (M. Errera and A. Forsberg, eds.) Vol. II, p. 231. 3cademic Press, New York, 1960. 4. GRWERT, R. R., AXD PHILLIPS, P. H., Arch. Bioclwn. Biophys. 30,217 (1951). 5. ELLXAS, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 6. BESESCH, R. H., BEKESCH, R., .+ZTDLARDY, H. A., J. Biol. Chem. 216,663 (1955). 7. DOIIAS, J. S., .~KD WOODWARD, G. E., J. Bid. Chem. 129,393 (1939). 8. ALEEXA~DER, P., BACQ, Z. M., COUSENH, S. F.,
r----
I’HK
9.
-------
JWI’I‘UHS
ELDJARN,
I,..
.IP;D
l+fIL,
A., 1. Niol. Chcm. 223,
341 (1956). 10.
.~~RFF:LDT.
S.,
Chcm.
!tcta
&and.
14,
1019
(1960). 11.
%jRBO,
12.
PIHL,
n..
dcta
AI.,
ELDJMS.
(‘hcrth.
12, 1990 (1958). BIWNR, ,J., J. Bid.
khnd.
I,..
A~I)
(‘hem. 227, 339 ( 1957). ielci~n. p&id. 62, 76 (1954). R. I,.. HEIFFER, M. H., AND IAIFIIEIT, 14. Mc~\I)T.
I-1. .J.. Satuw 16. VAS “ER iChR,
Dimerism
186, 312 (1960). c..
.\SD
of Chlorophyll
\‘.$I’
BEKKUM,
1).
I\'.,
a in Benzene’
The dimcrization of cl~lorophyll (c in conrcntrated ethanolic solutions has been suggested (1) on the basis of the difference spectrum between 3 X 10.’ and 10.” N solutions, and an analysis of activation spectra for fluorcr;ccsnre (2). While cstablishing a more definitive basis for earlirr similnl, suggest,ions (3, 4), thfbsr studies still do not provitkl direcat rridrncc of I he aggregated state or, more particularly, of t,licl degree of wggrrgation. In t,hi,~ communication we utilize a colligative propcrt: to demonstrate the dimerism of concentrated solut,ions of chlorophyll (I in henzenc and its increasing dissociation on dilution. The dimerism of c,arboxylic acids in nprotic solutions has been studied for many years (5), using cryoscopic, spectrophotomctri(*, and isopiestic: methods. A common reference monomer in thcsc studies has bcrn bc,nzil (dihcnzoyl), while benzoic acid hns been a frc)c!uent, example of an associating. 1 This research was supported in pat,t hy Nat,ional Sciencae I~oundation, (:rant So. G-75(33 ~cl~lorophyll). Journal Paper So. J-4371 of t,hca Iowa .igt~icultur:ll nntl Home Economics Kxpcriment Station, L1n~~s, Iowa. Pro,iec>t No. 1472. This is also Institute for Atomic Research Paper Ko. 1192.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Letters The Effect of Rodioprotective Nonprotein Sulfhydryl and
Agents Disulfide
98
(1962)
to the Editors
on Tissue Levels’
The radioprotective action of aminoethylthiosulfuric acid and aminoethplthiophosphate has been previously reported (1, 2), and the possibilit)y was considered that t,he protective action of these compounds was due to an in 2liz1o conversion of the thioesters to the corresponding sulfhydryl compound, cysteamine. The latter is a well-known mdioprotective compound. In order to test this hypot,hcsis the thiocsters now have been injected into mice and the nonprotein sulfhydryl and disulfide content of spleen, liver and blood has been dekrmined. Splern was chosen as an example of a radiosensitive tissue, liver represented a more radioresistant although metabolically very active tissue and blood was included for reasons drscribed later on. The in\-estigation was extended i o inrludc certain other protective and nonprotective compounds of interest in this conn&ion. The preparations of aminoethylthiosulfuric acid and nminoethylthiophosphate have been described previously (1. 2). Cystamine dihydrochloride was prepared from cystc‘nmine by oxidation with hpdrogm peroxide and recrystallizations from cthanol-hydrochloric arid. Other compounds were commercial products. The animals were female CBA-micr of 20 g. body weight. The compounds at dose levels reported (I-3) to give prot,ect,ion or, in case of thioglpc.olat,e and mrrcaptoethanol, no protection, WPI’P dissolved in saline and given hy intrappritoneal injection in a volume of 0.2 ml. J$%en lirrr and spleen were assayed, the animals were killed 10 min. after injection by immersion in an ethanolsolid rarbon dioxide mixture. Blood was analyzed in separate experiments. The samples in this case wcle obtained by heart puncturr also 10 min. after injection of the compound studied. Pooled tissue samples from 2-4 mice wpre then homogenized in ice-cooled 4% (w/l-) sulfosalirylic acid (lo-20 volumes per weight of tissue) in a glass Potter-Elvehjrm homogenizer. The homogenate was rentrifugcd and the sulfhydryl content of the, supernat,ant drtermined by a nitroprussidc mrlthod (4). In rasc of q&amine, aminoethylthiosulfuric arid, aminoethylthiophosphate, and nontreatcd controls deter‘This work has been supported by a grant from the Swedish Medical Rcsearc>h C’olmcil.
minations were also made by the dithiobisnitrohenzoic acid method (5), which had the advantage of giving stable absorbancy readings in contrast to the nitroprusside method. Satisfactory agreement (within 25%) was obtained always with the two methods. gttempts also were made to dctermine the sulfhydryl content by amperometric silver titration (6), but although glutathionc gave a sharp and reproducible end point, cystcamine did not, and Lhis method consequently was abandoned. Nonprotein disulfide compounds were determined on an aliquot of the sulfosalicylic acid extract tq electrolytic reduction (7) of the disulfitle to the corresponding sulfhydryl compound and determination of tho latter with nitroprusside. The thioesters were, howevrr, found to interferc7 in this determination as they also were reduced c>lcrtrolytically to the rorrrsponding sulfhydryl rompound. It was found (Table I) that injection of all the sulfur-containing compounds gave increased nonprotein sulfhydryl levels in the tissues studied. The differences were statistically significant (p < 0.05). It is rvident, from Table I that as the increases in liver and spleen were larger than thosr in blood, the increasrd values in liver and spleen could not be attrihutrd to the blood content of these tissues. It also was found that, the pharmacological a&-e amine serotonin (5-hydroxytrypktmine) which does not contain “potential” sulfhpdrpl groups, gave a slight, hut significant inrrease of sulfhydryl l~lvrl in spleen and liver (Table I). The effect of the compounds studied on t.hr nonprotein disulfide compounds of spleen and liver is shown in Table II. In this case no or J-cry small increases were ohservcd. Only in the liver after cystamine, mrrcaptoethanol, and aminoethylthiosulfuric acid and in the spleen nftrr t,hc, lust-mcntioned compound were significant incrclascs notr,tl (p < 0.05). The increased values observed after injection of the thioestcr may, howcvclr, hr due t,o the pre$mce of the parent compound itself, whirh intcrfrres with the disulfidc detrrmination, ii.5 *jrr>viously mentioned. Th(> results obtained in the presrnt investigation are of some interest in connection with the mrchanism of action of the radioprotcctive drugs. It has been suggested for instance that the radioprotcctive sulfhpdryl compounds owe their effert, to thrir ntA1it.v to react with radiation-induced free radicsalp
LETTERS
TO THE TABLE
Compound injected
fpmoles/g.)
+
0.14
(8)
200 200 360
6.31 + 0.18 6.3“ f 0.12 5.24 f 0.2i
(5) (5) (6)
400
5.89 i
(6)
265 150 50
5.69 f 0.073 (4) 6.38 f 0.13 (4) 3.88 IIZ 0.0% (6)
u Values given as mean f
standard
error.
0.20
Figures TBBLE
DISI-LFIDE
Compound injected
Cysteamine HCI Cystamine. 2HCl Aminoethylthiosulfuric acid Sodium aminoethylthiophosphate Sodium thioglycolate Mercaptoethanol Serotonin-creatinine sulfate a Values
Liver
Spleen 3.40
~ONI'ROTEIN
I
Sulfhydryl content
DOW bllg./kg.)
Cystenmine. HCI Cystamine. 2HCl Arnirloethvlthioslllfuric acid Sodium aminoetjhythiophosphat,e Podium thioglycolate Mercaptoethanol Serotonincreatinine sulfat,e
given as mean & standard
343
EDITORS
r,EVELS
7.56
i
Blood
0.25
(8)
0.56
0.18 0.49 0.09
(5) (5) (6)
1.19 i 0.i-f
9.65 zk 0.23
(6)
1 .21 + 0.061
(4)
1.45 f
(4)
10.3 f 9.5i f X.88 f
8.29 It 0.18 (1) 10.2 It 0.15 (4) 8.29 zk 0.22 (6)
in parentheses
indicate
number
f
0.04
(4)
0.098
(1)
zt 0.049
0.043
of determinat~ions.
II
IN Tmsc-ES
OF TREATEI)
.4x1) COSTROL
~~I(W
DOS+? (mg.jkg.j
0.17 0.43 0.33 0.73 0.26 0.06 0.15 -0.01
200 200 360 400 265 150 5!) error.
Figures
(8). The present investigation showed that both the protective cysteamine and the nonprotective thioglpcolate and mercaptoethanol gave similar increases of the sulfhydryl concentration in the spleen. However, protective and nonprotective sulfhydryl compounds seem to be equally effective for inactivation of free radicals (3) and (intracellular distribution phenomena being disregarded) the present results thus do not support the “radicalscavenger” hypothesis. Other theories, such as the “mixed-disulfide” hypothesis (9) which take into account the difference in chemical reactivities of the different sulfhydryl compounds seem to be in better agreement with the results of the present investigation. Concerning the mechanism of action of the thioesters studied (aminoethylthiosulfuric acid and aminoethylthiophosphate) the present results suggest, that these compounds are converted in viva
f 0.063 f 0.15 f 0.11 z!c 0.086 2~ 0.13 f 0.033 Zt 0.13 + 0.039
in parentheses
(8) (5) (5) (6) (6) (4) (1) (6)
indicate
0.03 0.09 0.38 0.70 0.06 0.02 0.32 -0.01 number
f f + f + f + i
0 023 (8) 0.025 (5) 0.11 (5) 0.080 (6) 0.016 (6) 0.011 (4) 0.033 (4) 0.017 (6)
of tlrterminations.
to cysteamine, lvhich then may be the effective protective agent. These conversions are probably enaymic, and an enzymic hydrolysis of the thiophosphate has in fact been demonstrat,ed by Akerfeldt (10). In case of the thiosulfate ester, however, previous in zrilro studies (11) gave no evidence for an enzymic hydrolysis, and further experiments (unpublished) showed that if the compound was incubated with a mouse liver homogenate, no increase, but on the contrary a decrease of the nonprotein sulfhydryl concentration in the homogenate was observed. This effect is explained by the fact that, the thiosulfat)e ester reacts spontaneously with glutathione (11) to give the mixed disulfide of cpsteamine and glutathione. The latter may, however, be expected to be reduced to the free thiols through the action of glutathione reductase according to the mechanism elucidated by Pihl et al. (12). The existence of such a reac-
LETTERS
341
TO
tion was supported by the fact that, when a systcm containing liver homogenate and thiosulfate ester was supplemented with TPS plus glucose-6phosphate (which gives TPKH due t,o the presence of glucose-5-phosphntc dchydrogenase in the homogenate), an increase of nonprotein sulfhydryl groups was obscr\.rd. It is of interest that all the sulfur-containing protective compounds studied in this investigation, being either thiols, disulfides, or thioesters, gave increased nonprot,ein sulfhydryl levels in the tissues but only slight or no increases in disulfide levels. The rapid reduction of cystamine in viva to cysteamine has been noted previously by others (13, 14). If the radioprotective action of the sulfur-containing agents is due to a formation of mixed disulfides with tissue constit,uents, as suggcsted by Eldjarn and Pihl (91, the present results indicate that the mixed disulfides are formed by interaction of radiosmsitive disulfidc bonds in thr tissue and the protecti\-c agent. The latter would he a sulfhydryl compound from the beginning or converted to such a cornl)ound in the body. The sensitivity of disulfide bond,? i o ionizing radiation has in fact been notcad (3, 15). Finally, the fact that scrotonin also caused a slight increase in the sulfhydryl content of the spleen merits some discussion. The excellent work of van der Meer and van Bekkum (16) has demonstrated that the radioprotective effect of serotonin can be attribut,ed to s diminished “oxygen cart,” as serotonin caus(xs a pronoun& tissue hypoxia in the spleen and bone marrow. It is possible, however. that the incrrasc in nonprotein sulfhydryl compounds obsclr\-ed in t,he present investigation contributc>s to the r:tdioprotecti\-e rffeet of serotonin. REFERENCES 1. HOLMBERG, B., ASD S~RBO, B., Nature 183, 832 (1959). 2. H.~NSEN, B., ASD SGRBO, B., Acta Radial. 56, 141 (1961). 3. ELDJARK. L., .~ND PIHI,. A., In “Mechanisms in Radiobiology” (M. Errera and A. Forsberg, eds.) Vol. II, p. 231. 3cademic Press, New York, 1960. 4. GRWERT, R. R., AXD PHILLIPS, P. H., Arch. Bioclwn. Biophys. 30,217 (1951). 5. ELLXAS, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 6. BESESCH, R. H., BEKESCH, R., .+ZTDLARDY, H. A., J. Biol. Chem. 216,663 (1955). 7. DOIIAS, J. S., .~KD WOODWARD, G. E., J. Bid. Chem. 129,393 (1939). 8. ALEEXA~DER, P., BACQ, Z. M., COUSENH, S. F.,
r----
I’HK
9.
-------
JWI’I‘UHS
ELDJARN,
I,..
.IP;D
l+fIL,
A., 1. Niol. Chcm. 223,
341 (1956). 10.
.~~RFF:LDT.
S.,
Chcm.
!tcta
&and.
14,
1019
(1960). 11.
%jRBO,
12.
PIHL,
n..
dcta
AI.,
ELDJMS.
(‘hcrth.
12, 1990 (1958). BIWNR, ,J., J. Bid.
khnd.
I,..
A~I)
(‘hem. 227, 339 ( 1957). ielci~n. p&id. 62, 76 (1954). R. I,.. HEIFFER, M. H., AND IAIFIIEIT, 14. Mc~\I)T.
I-1. .J.. Satuw 16. VAS “ER iChR,
Dimerism
186, 312 (1960). c..
.\SD
of Chlorophyll
\‘.$I’
BEKKUM,
1).
I\'.,
a in Benzene’
The dimcrization of cl~lorophyll (c in conrcntrated ethanolic solutions has been suggested (1) on the basis of the difference spectrum between 3 X 10.’ and 10.” N solutions, and an analysis of activation spectra for fluorcr;ccsnre (2). While cstablishing a more definitive basis for earlirr similnl, suggest,ions (3, 4), thfbsr studies still do not provitkl direcat rridrncc of I he aggregated state or, more particularly, of t,licl degree of wggrrgation. In t,hi,~ communication we utilize a colligative propcrt: to demonstrate the dimerism of concentrated solut,ions of chlorophyll (I in henzenc and its increasing dissociation on dilution. The dimerism of c,arboxylic acids in nprotic solutions has been studied for many years (5), using cryoscopic, spectrophotomctri(*, and isopiestic: methods. A common reference monomer in thcsc studies has bcrn bc,nzil (dihcnzoyl), while benzoic acid hns been a frc)c!uent, example of an associating. 1 This research was supported in pat,t hy Nat,ional Sciencae I~oundation, (:rant So. G-75(33 ~cl~lorophyll). Journal Paper So. J-4371 of t,hca Iowa .igt~icultur:ll nntl Home Economics Kxpcriment Station, L1n~~s, Iowa. Pro,iec>t No. 1472. This is also Institute for Atomic Research Paper Ko. 1192.