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Reaction of gossypol with free ϵ-amino groups of lysine in proteins
ARCHIVES
OF
BIOCHEMISTRY
Reaction
AND
BIOPHYSICS
13&134
(19%)
of Gossypol with Free ~-Amino Lysine in Proteins
Edith J. Conkerton From the Southern
81,
Groups of
and Vernon L. Frampton
Regional Research Laboratory’ and the National Cottoweed Association Fellowship, New Orlea.ns, Lowisiana Received
September
Products
15, 1958
INTRODUCTION Evidence is presented in this communication for the addition of gossypol (2,2’-bi-1,6,7-trihydroxy-3-methyl-5-isopropyl-8-aldehydonaphthyl) to the free c-amino groups of lysine in bovine serum albumin, egg albumin, p-lactoglobulin, and cottonseed globulin, presumably to yield Schiff bases. The reaction of gossypol, a polyphenolic pigment indigenous in cottonseed, with the free e-amino groups of lysine in cottonseed proteins is of considerable domestic importance because the three million tons of cottonseed meal which are produced annually in t,he United States constitute a major source of lysine in the feeding of monoga&ric animals. According t,o Baliga and Lyman (I), the availability of lysine, as determined by ratfeeding tests, was reduced when cottonseed meals were allowed to react with gossypol. The demonstration of a reaction between gossypol and the free c-amino groups of lysine in proteins was achieved through the use of 2,4dinitrofluorobenzene; the number of e-amino groups that are free to react with 2,4-dintrofluorobenzene is reduced when the proteins are exposed 60 gossypol. Carpenter et al. (2) reported a correlation between the “available” lysine of animal proteins, as determined through the use of 2,4dinit,rofluorobenzene, and the nut#ritional quality of the proteins. They reported, however, that the procedure was not applicable to proteins of plant origin. In the work reported here, t,he presence of relatively large quantities of 2,4dinitrophenol was noted in hydrolyzates of plant proteins that had been treated with 2,4dinitrofluorobenzene and hydrolyzed with 6 N hydrochloric acid. The presence of t,he phenol added a complication to the spectrophotometric determination of e-2,4dinitrophenyllysine, as it is difficult to make a quantitative separation of these two yellow substances. 1 A laboratory of the Southern Utilizat,ion Research and Development. ilgricult.ural Research Service, U. S. Department of Agriculture. 130
Division,
REACTION
OF
131
GOSSYPOL
The difficulty was circumvented by taking advantage of the fact that the absorption behavior of 2 ,Qdinitrophenol is pH dependent, i.e., the absorption maximum at 360 rnp for an alkaline medium disappears and the absorption maximum at 260 rnl.r is greatly accentuat,ed when the solution is acidified, whereas the absorption behavior for the lysine derivative is not pH dependent. Because the absorptivities of these two substances are additive under the experimental conditions used, an estimation of the quantity of e-2 ,i-dinitrophenyllysine present in the mixture can be obtained by observing the absorption at t.wo different pH levels, and correcting for the quantity of the phenol present. This procedure is applicable to both animal and plant proteins. 2 ,+LDinitrophenyl derivatives of the other amino acids did not interfere with t,he determination of E-2,4-dinit,rophenyllysine, since those which appeared in the hydrolyzat,es were removed quantitatively on extraction with ethyl ether. It was demonstrated, through the use of internal and external standards in a paper chromatographic study, that the only yellowcolored products present in the ether-washed acid hydrolyzates of the 2, d-dinitrophenyl derivatives of prot,eins were 2,4dinitrophenol and e-2,4-dinitrophenyllysine. Therefore, the only correction required for the materials studied was that for the phenol. The values for lysine calculated from the e-amino group data for the proteins studied are in subst,antial agreement wit#h the total lysine values determined by other methods (Table I). The data included in Table I indicate the extent to which the number of free t-amino groups of lysine is diminished when the proteins are exposed to gossypol under these experimental conditions. It was also noted that the analyses of prot,eins which were exposed to the same experimental conditions in the absence of gossypol showed no change in the number of free E-amino groups of lysine. From t,he data in Table I, it is apparent that the extent of the reaction of gossypol with the proteins is influenced by the pH of the medium and that the reduction of t,he number of free e-amino groups of lysine in the proteins is greater at t,he higher pH value. h standard deviation for the method of 0.03 g. lysine/l6 g. nitrogen was calculated for a series of 30 determinations. Hence, it is apparent t,hat, the experimental errors encountered are a small fraction of the differences observed for the values for the t-amino groups of lysine before and after reaction of the proteins with gossypol. EXPERIMENTaL
Protehs. commercial
The bovine origin.
serum
albumin,
&lactoglobulin,
and egg albumin
were of
132
CONKERTON
AND
TABLE Reaction
of Gossypol
with Free e-.-imino
FRAMPTON
I Groups of Lysine Free c-amino
Lysine
contenta
in Proteins lysine
content*
Gossypol-protein Original
complex
material PR 6
g/16
Bovine serum albumin fl-Lactoglobulin Egg albumin Cottonseed
globulin
g. N
8.412.& 10.2-13. 3d 11.3e 5.6-7.01 6.6e 3.59
g.116 g. N
g/16
g. N
PR 8 g/ill
g. N
11.9 12.5
8.3 10.2
6.7
4.1
2.7
3.5
3.0
1.2
a As reported in the literature. 6 As determined by the use of 2,4dinitrofluorobenzene, this investigation. L , “Amino Acid Handbook,” p. 252. Charles C c R. J. Block and K. W. Weiqs Thomas, Springfield, Ill., 1956. d Ibid. p. 268. 6 F. Sanger, Biochem. Sot. Symposium No. 3, 29 (1950). JR. J. Block and K. 11’. Weiss, “Amino Acid Handbook,” p. 258. Charles C Thomas, Springfield, Ill., 1956. 0 Private communication, W. H. Martinez. This value was obt,ained t,hrough the use of the ion-exchange procedure of S. Moore and W. H. Stein, J. Biol. Chem. 192. 663 (1951).
The cottonseed globulin was prepared by exhaustive extraction of flaked kernels of prime cottonseed successively with commercial hexane, ethyl met,hyl ketone, and 0.5 M aqueous NaCl. The saline extracts were combined and dialyzed against cold, running, distilled water. The dialyzate was then lyophilized. 2,.&DinitrophenoZ. The 2,4dinitrophenol, m.p. ill-111”, was obtained through purification of a technical grade by recrystallization. e-2,4-Dinitrophenyllysine. The preparation of this material, m.p. 187-8”, was carried out in accordance with the directions given by Porter (3). Gossypol. The material used in this study was prepared from prime cottonseed in accordance with the directions given by King and Thurber (4). 2,4-DinitroJEuorobenrene. This reagent was of commercial origin. Gossypol-Protein Complezes. The gossypol-protein complexes were prepared in solution at approximately pH’s 6 and 8, by dispersing or dissolving 200 mg. protein in 50 ml. water or in 50 ml. of 10% aqueous NaHC03 solution. Fifty milliliters of ethanol, containing sufficient gossypol t,o react with all free amino groups of lysine in the protein on a mole to mole basis, were added to t.he aqueous solut.ion. The resultant mixtures were gently agitated on a mechanical shaker for 24 hr. at room temperature. They were then filtered, and the precipitat,ed gossypol-protein complexes were washed, first with water and then with ethyl ether, until the washings were colorless. The yellow products were ground to powders in a glass mortar and stored in a desiccator over anhydrous CaSOl .
REACTION
OF GOSSYPOL
133
Methods Preparation of 2,4-Dinitrophenyl Proteins. Except for minor modifications, the 2,4-dinitrophenyl proteins were prepared by the method employed by Banger (5). Three lOU-309mg. samples of the protein or gossypol-protein complex were weighed into 5O&ml. round-bottom flasks. Ten milliliters of 10% aqueous NaHCC3 and a few glass beads were added to each flask. The resultant solutions were thoroughly mixed and allowed to stand at room temperature for 10 min. Then 13 ml. et.hanol containing 0.45 g. 2,4-dinitrofluorobenzene was added to two of the flasks. -411of the llasks were shaken slowly on a mechanical shaker for 2 hr. at room temperature. The mixture was then concentrated nearly to dryness at room temperature under a stream of air. Unchanged 2,4-dinitrofluorobenzene and some of the 2,4-dinitrophenol produced by side reactions were extracted from the mixtures with four successive 50.ml. portions of ethyl ether. The ether was mixed thoroughly with the semisolid mass, and the two phases were allowed to separate completely before the ether was decanted. Final traces of ether were removed by an air stream passed over the semisolid mass. The third sample, t.o which no 2,4-dinitrofluorobenzene was added, served as a blank. Hydrolysis of 2,Q-Dinitrophenyl Proteins. The samples and the blank were hydrolyzed by refluxing them for 16 hr. wit.h 50 ml. of constant-boiling HCl. The hydrolyzates were cooled, filtered through medium por0sit.y sintered-glass funnels, and washed with water. The filtrates and washings in each case were combined and diluted with wat.er to 100 ml. Determination of c-2,4-Dinitroph.enyllysine. A lo-ml. aliquot of each hydrolyzate was extracted in a separatory funnel with four successive 59ml. portions of ethyl ether to remove interfering 2,4-dinitrophenyl amino acids, which are ether soluble, and additional quantities of 2,4-dinitrophenol. It was only necessary to wash the blank with two portions of ether. The ether-washed hydrolyzates were diluted to 25 ml., in a volumetric flask, with water, after t.he ether in solution had been removed. Duplicate aliquots of each solut.ion (3-5 ml., depending on the intensity of the yellow color) were placed in 25.ml. volumetric flasks. One of each was diluted to volume with 1 K HCl and the other with 10yc, aqueous NaHC03. The absorbancies of these final solutions were determined at 360 rnp on a Beckman model B spectrophotometer2 using the corresponding blank as the reference solution. The concentrat,ion of 2,&dinitrophenol (DNP), in mg./ml., was calculated by: (DNP)
= K (-4, -
92),
where d, and Az are the absorbancies of the sodium bicarbonate and acid solutions, respectively, and K is 0.0158, the reciprocal of the difference between the absorptivities at 360 rnr of 2,4-dinitrophenol in alkaline and acid solutions, as determined experimentally. The concentration of e-2,4-dinitrophenyllysine (s-DNP-lysine), in mg./ml., was then calculated by: (c-DNP-lysine) where K1 is 7i.4, the absorptivity tivity of s-2,4-dinitrophenyllgsine,
A, - K,(DNP) = ~ KZ
of 2,4-dinitrophenol, and Kz is 46.9, the absorpboth at 360 mp in sodium bicarbonate solution.
2 Trade names are given as part of the exact experimental conditions an endorsement of the product over those of other manufacturers.
and not as
134
CONKERTON
AND FRAMPTON
SUMMaRY
The number of e-amino groups of lysine in proteins which are free to react with 2,&dinitrofluorobenzene is reduced when the proteins are exposed to reaction with gossypol. The reduction in reactive c-amino groups of lysine is increased when the pH of the reaction mixture is increased. A procedure for estimation of t,he e-amino groups is described. REFERENCES 1. BALIGA, B. P., AND LYMAN, C. M., J. Am. Oil Chemists Sot. 34, 21 (1957). 2. CARPENTER, K. .J.,ELLIHGER, G.M.,Muno, J. I., AND RoLF,E.J., Brit.J.Nutrifion 11, 162 (1957). 3. PORTER, R. R., Meth.ods in Med. Research 3, 259 (1950). 4. KING, W. H., AND THURBER, F. H., J. .-lwt. Oil Chemists Sot. 30, 70 (1953). 5. SANGER, F., Biochem. J. 39, 507 (1945).
OF
BIOCHEMISTRY
Reaction
AND
BIOPHYSICS
13&134
(19%)
of Gossypol with Free ~-Amino Lysine in Proteins
Edith J. Conkerton From the Southern
81,
Groups of
and Vernon L. Frampton
Regional Research Laboratory’ and the National Cottoweed Association Fellowship, New Orlea.ns, Lowisiana Received
September
Products
15, 1958
INTRODUCTION Evidence is presented in this communication for the addition of gossypol (2,2’-bi-1,6,7-trihydroxy-3-methyl-5-isopropyl-8-aldehydonaphthyl) to the free c-amino groups of lysine in bovine serum albumin, egg albumin, p-lactoglobulin, and cottonseed globulin, presumably to yield Schiff bases. The reaction of gossypol, a polyphenolic pigment indigenous in cottonseed, with the free e-amino groups of lysine in cottonseed proteins is of considerable domestic importance because the three million tons of cottonseed meal which are produced annually in t,he United States constitute a major source of lysine in the feeding of monoga&ric animals. According t,o Baliga and Lyman (I), the availability of lysine, as determined by ratfeeding tests, was reduced when cottonseed meals were allowed to react with gossypol. The demonstration of a reaction between gossypol and the free c-amino groups of lysine in proteins was achieved through the use of 2,4dinitrofluorobenzene; the number of e-amino groups that are free to react with 2,4-dintrofluorobenzene is reduced when the proteins are exposed 60 gossypol. Carpenter et al. (2) reported a correlation between the “available” lysine of animal proteins, as determined through the use of 2,4dinit,rofluorobenzene, and the nut#ritional quality of the proteins. They reported, however, that the procedure was not applicable to proteins of plant origin. In the work reported here, t,he presence of relatively large quantities of 2,4dinitrophenol was noted in hydrolyzates of plant proteins that had been treated with 2,4dinitrofluorobenzene and hydrolyzed with 6 N hydrochloric acid. The presence of t,he phenol added a complication to the spectrophotometric determination of e-2,4dinitrophenyllysine, as it is difficult to make a quantitative separation of these two yellow substances. 1 A laboratory of the Southern Utilizat,ion Research and Development. ilgricult.ural Research Service, U. S. Department of Agriculture. 130
Division,
REACTION
OF
131
GOSSYPOL
The difficulty was circumvented by taking advantage of the fact that the absorption behavior of 2 ,Qdinitrophenol is pH dependent, i.e., the absorption maximum at 360 rnp for an alkaline medium disappears and the absorption maximum at 260 rnl.r is greatly accentuat,ed when the solution is acidified, whereas the absorption behavior for the lysine derivative is not pH dependent. Because the absorptivities of these two substances are additive under the experimental conditions used, an estimation of the quantity of e-2 ,i-dinitrophenyllysine present in the mixture can be obtained by observing the absorption at t.wo different pH levels, and correcting for the quantity of the phenol present. This procedure is applicable to both animal and plant proteins. 2 ,+LDinitrophenyl derivatives of the other amino acids did not interfere with t,he determination of E-2,4-dinit,rophenyllysine, since those which appeared in the hydrolyzat,es were removed quantitatively on extraction with ethyl ether. It was demonstrated, through the use of internal and external standards in a paper chromatographic study, that the only yellowcolored products present in the ether-washed acid hydrolyzates of the 2, d-dinitrophenyl derivatives of prot,eins were 2,4dinitrophenol and e-2,4-dinitrophenyllysine. Therefore, the only correction required for the materials studied was that for the phenol. The values for lysine calculated from the e-amino group data for the proteins studied are in subst,antial agreement wit#h the total lysine values determined by other methods (Table I). The data included in Table I indicate the extent to which the number of free t-amino groups of lysine is diminished when the proteins are exposed to gossypol under these experimental conditions. It was also noted that the analyses of prot,eins which were exposed to the same experimental conditions in the absence of gossypol showed no change in the number of free E-amino groups of lysine. From t,he data in Table I, it is apparent that the extent of the reaction of gossypol with the proteins is influenced by the pH of the medium and that the reduction of t,he number of free e-amino groups of lysine in the proteins is greater at t,he higher pH value. h standard deviation for the method of 0.03 g. lysine/l6 g. nitrogen was calculated for a series of 30 determinations. Hence, it is apparent t,hat, the experimental errors encountered are a small fraction of the differences observed for the values for the t-amino groups of lysine before and after reaction of the proteins with gossypol. EXPERIMENTaL
Protehs. commercial
The bovine origin.
serum
albumin,
&lactoglobulin,
and egg albumin
were of
132
CONKERTON
AND
TABLE Reaction
of Gossypol
with Free e-.-imino
FRAMPTON
I Groups of Lysine Free c-amino
Lysine
contenta
in Proteins lysine
content*
Gossypol-protein Original
complex
material PR 6
g/16
Bovine serum albumin fl-Lactoglobulin Egg albumin Cottonseed
globulin
g. N
8.412.& 10.2-13. 3d 11.3e 5.6-7.01 6.6e 3.59
g.116 g. N
g/16
g. N
PR 8 g/ill
g. N
11.9 12.5
8.3 10.2
6.7
4.1
2.7
3.5
3.0
1.2
a As reported in the literature. 6 As determined by the use of 2,4dinitrofluorobenzene, this investigation. L , “Amino Acid Handbook,” p. 252. Charles C c R. J. Block and K. W. Weiqs Thomas, Springfield, Ill., 1956. d Ibid. p. 268. 6 F. Sanger, Biochem. Sot. Symposium No. 3, 29 (1950). JR. J. Block and K. 11’. Weiss, “Amino Acid Handbook,” p. 258. Charles C Thomas, Springfield, Ill., 1956. 0 Private communication, W. H. Martinez. This value was obt,ained t,hrough the use of the ion-exchange procedure of S. Moore and W. H. Stein, J. Biol. Chem. 192. 663 (1951).
The cottonseed globulin was prepared by exhaustive extraction of flaked kernels of prime cottonseed successively with commercial hexane, ethyl met,hyl ketone, and 0.5 M aqueous NaCl. The saline extracts were combined and dialyzed against cold, running, distilled water. The dialyzate was then lyophilized. 2,.&DinitrophenoZ. The 2,4dinitrophenol, m.p. ill-111”, was obtained through purification of a technical grade by recrystallization. e-2,4-Dinitrophenyllysine. The preparation of this material, m.p. 187-8”, was carried out in accordance with the directions given by Porter (3). Gossypol. The material used in this study was prepared from prime cottonseed in accordance with the directions given by King and Thurber (4). 2,4-DinitroJEuorobenrene. This reagent was of commercial origin. Gossypol-Protein Complezes. The gossypol-protein complexes were prepared in solution at approximately pH’s 6 and 8, by dispersing or dissolving 200 mg. protein in 50 ml. water or in 50 ml. of 10% aqueous NaHC03 solution. Fifty milliliters of ethanol, containing sufficient gossypol t,o react with all free amino groups of lysine in the protein on a mole to mole basis, were added to t.he aqueous solut.ion. The resultant mixtures were gently agitated on a mechanical shaker for 24 hr. at room temperature. They were then filtered, and the precipitat,ed gossypol-protein complexes were washed, first with water and then with ethyl ether, until the washings were colorless. The yellow products were ground to powders in a glass mortar and stored in a desiccator over anhydrous CaSOl .
REACTION
OF GOSSYPOL
133
Methods Preparation of 2,4-Dinitrophenyl Proteins. Except for minor modifications, the 2,4-dinitrophenyl proteins were prepared by the method employed by Banger (5). Three lOU-309mg. samples of the protein or gossypol-protein complex were weighed into 5O&ml. round-bottom flasks. Ten milliliters of 10% aqueous NaHCC3 and a few glass beads were added to each flask. The resultant solutions were thoroughly mixed and allowed to stand at room temperature for 10 min. Then 13 ml. et.hanol containing 0.45 g. 2,4-dinitrofluorobenzene was added to two of the flasks. -411of the llasks were shaken slowly on a mechanical shaker for 2 hr. at room temperature. The mixture was then concentrated nearly to dryness at room temperature under a stream of air. Unchanged 2,4-dinitrofluorobenzene and some of the 2,4-dinitrophenol produced by side reactions were extracted from the mixtures with four successive 50.ml. portions of ethyl ether. The ether was mixed thoroughly with the semisolid mass, and the two phases were allowed to separate completely before the ether was decanted. Final traces of ether were removed by an air stream passed over the semisolid mass. The third sample, t.o which no 2,4-dinitrofluorobenzene was added, served as a blank. Hydrolysis of 2,Q-Dinitrophenyl Proteins. The samples and the blank were hydrolyzed by refluxing them for 16 hr. wit.h 50 ml. of constant-boiling HCl. The hydrolyzates were cooled, filtered through medium por0sit.y sintered-glass funnels, and washed with water. The filtrates and washings in each case were combined and diluted with wat.er to 100 ml. Determination of c-2,4-Dinitroph.enyllysine. A lo-ml. aliquot of each hydrolyzate was extracted in a separatory funnel with four successive 59ml. portions of ethyl ether to remove interfering 2,4-dinitrophenyl amino acids, which are ether soluble, and additional quantities of 2,4-dinitrophenol. It was only necessary to wash the blank with two portions of ether. The ether-washed hydrolyzates were diluted to 25 ml., in a volumetric flask, with water, after t.he ether in solution had been removed. Duplicate aliquots of each solut.ion (3-5 ml., depending on the intensity of the yellow color) were placed in 25.ml. volumetric flasks. One of each was diluted to volume with 1 K HCl and the other with 10yc, aqueous NaHC03. The absorbancies of these final solutions were determined at 360 rnp on a Beckman model B spectrophotometer2 using the corresponding blank as the reference solution. The concentrat,ion of 2,&dinitrophenol (DNP), in mg./ml., was calculated by: (DNP)
= K (-4, -
92),
where d, and Az are the absorbancies of the sodium bicarbonate and acid solutions, respectively, and K is 0.0158, the reciprocal of the difference between the absorptivities at 360 rnr of 2,4-dinitrophenol in alkaline and acid solutions, as determined experimentally. The concentration of e-2,4-dinitrophenyllysine (s-DNP-lysine), in mg./ml., was then calculated by: (c-DNP-lysine) where K1 is 7i.4, the absorptivity tivity of s-2,4-dinitrophenyllgsine,
A, - K,(DNP) = ~ KZ
of 2,4-dinitrophenol, and Kz is 46.9, the absorpboth at 360 mp in sodium bicarbonate solution.
2 Trade names are given as part of the exact experimental conditions an endorsement of the product over those of other manufacturers.
and not as
134
CONKERTON
AND FRAMPTON
SUMMaRY
The number of e-amino groups of lysine in proteins which are free to react with 2,&dinitrofluorobenzene is reduced when the proteins are exposed to reaction with gossypol. The reduction in reactive c-amino groups of lysine is increased when the pH of the reaction mixture is increased. A procedure for estimation of t,he e-amino groups is described. REFERENCES 1. BALIGA, B. P., AND LYMAN, C. M., J. Am. Oil Chemists Sot. 34, 21 (1957). 2. CARPENTER, K. .J.,ELLIHGER, G.M.,Muno, J. I., AND RoLF,E.J., Brit.J.Nutrifion 11, 162 (1957). 3. PORTER, R. R., Meth.ods in Med. Research 3, 259 (1950). 4. KING, W. H., AND THURBER, F. H., J. .-lwt. Oil Chemists Sot. 30, 70 (1953). 5. SANGER, F., Biochem. J. 39, 507 (1945).