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Biologically active thiocarbamyl derivatives of insulin
Biologically
Active Thiocarbamyl
Derivatives
of Insulins
Emil Kaiser,2 L. C. Maxwell, W. A. Landmann and Robert Hubata From the Armour
Laboratories,
Chicago,
Illinois
Received August 18, 1952
The functional groups responsible for the biological activity of insulin have been studied by a number of investigators. A detailed discussion of this subject was recently published by J. and H. Fraenkel-Conrat (1). According to these authors, several groups, among them the free amino groups, may be largely involved in chemical reactions without any apparent loss of insulin activity. To broaden t,he study of the correlation between insulin activity and the number of free amino groups in the insulin molecule, the isothiocyanate reaction of Edman (2, 3) was applied to insulin. The use of the Edman procedure for the determination of the amino acid sequence in insulin was reported recently (4), but the biological activity of the t.hiocarbamyl derivatives was not studied. The react,ion between peptides and phenyl isothiocyanate was carried out by Edman in pyridinewater mixt,ures. Application of this procedure to insulin yielded biologically inactive products in our hands. Therefore it was attempted to use aqueous buffer solutions instead of pyridinewater mixtures. The insulin derivatives prepared in buffer solutions were biologically active when tested by the mouse convulsion assay. RESULTS
When phenyl isothiocyanate was added to a solution of insulin in a phosphate buffer at pH 7.67, a precipitate was slowly formed. The amount of the precipitated insulin depended upon the reaction time, and 1 Presented before the Division of Biological Chemistry, American Chemical Society, Milwaukee, March 31, 1952. * Present address: The Bio-l’rocess Co., Division of Armour h Co., .Joliet, 111. 94
THIOCARBAMYL
DERIVATIVES
OF
95
INSULIN
upon the weight ratio between insulin and phenyl isothiocyanate (Table I). When 10 parts of phenyl isothiocyanate per 1 part of insulin was used, corresponding to 710 moles of phenyl isothiocyanate/104 g. insulin, a reaction time of 24 hr. was needed to precipitate the insulin completely at temperatures of 15-20”. Seventy-one moles of phenyl isothiocyanate/104 g. insuli21, 1 part of phenyl isothiocyanat)e per 1 part of insulin, yielded only 60% of insulin precipitate within 24 hr. In these produck about 4(t-50y0 of the amino groups remained unreacted. The TABLE Free Amino
Phenyl isothiocyam&/l0’ g. insulin
710 710 710 71 71 71 14 0
Nitrogen
Reaction
time
I
and Biological Activity Insulin Derivatives Recovery of insulin in ppt.”
of Phenylthiocnrbamyl
Free NHz
groupsb
hr.
%
%
2 6 24 2 6 24 24 0
22 44 90 25 40 53 35
54 44 44 49 38 41 65 100
Biological
activityC
5
57 48 52 54 44 47 80 100
f f z!z f f f f f
8 7
8 8 7 7 12 10
a Averages of three determinations. b Averages of three duplicate determinations. c Biological activity was measured in the mouse convulsion Ii. S. P. reference standard.
test against
the
insulin activity, determined according to the mouse convulsion test, was about 50% of the original activity. When 14 moles of phenyl isothiocyanate were used for lo4 g. insulin, 1 part of phenyl isothiocyanate per 5 parts of insulin, the yield of the precipitate amounted to 35% only. This preparation had 65y0 of the free amino groups unchanged and about 80% of the original activity was retained. All of the phenylthiocarbamyl insulin precipitates showed prolonged blood sugar-lowering activity, similar to that of the neutralprotamine-of-Hagedorn (NPH) insulin, when tested in fasted rabbits. The phenylthiocarbamyl insulin precipitates were isolated from the buffer solutions by centrifugation in the presence of Ohe unreacted phenyl isothiocyanate. When the phenyl isothiocyanate was extracted
96
KAISER,
MAXWELL,
LANDMANN
AND
HUBATA
with ether before centrifugation, the precipitate dissolved in the buffer solution. The phenylthiocarbamyl insulin could be separated from the buffer by dialysis and subsequent freeze-drying of the dialyzed solution. These products were water-soluble, yielding a solution of pH 6.9-7.0. The dependence of the formation of an insulin precipitate at pH 7.67 upon the buffer-phenyl isothiocyanate system was further demonstrated when ally1 isothiocyanate was used instead of phenyl isothiocyanate. Ten parts of ally1 isothiocyanate per 1 part of insulin, 990 moles of ally1 isothiocyanate/104 g. insulin, did not precipitate an insulin
The Influence Biological Reaction
time
TABLE II of Reaction Time on the Free Amino GTOUPS and the Activity of Allylthiocarbamyl Insulin Derivatives Insulin
recovered as ppt.”
hr.
0
1 2 3 6 24
% -
85-90 85-90 85-90 85-90 85-90
Free NH,
group8
Biological
% 100
73 75 71 57 50
activityC %
loo
f
83 f 70 f 84 f 68 f 55f
10 12 10 13 10 8
DAverages of three determinations. b Averages of three duplicate determinations. c Biological activity was measured in the mouse convulsion test against the U. S. P. reference standard.
derivative from a phosphate buffer solution at pH 7.67. The allylthiocarbamyl insulin precipitated when the pH was adjusted to 5.5. The initial reaction rate of insulin with ally1 isothiocyanate was somewhat slower than with phenyl isothiocyanate (Tables I and II). But after 24 hr. both the ally1 and the phenyl products had approximately the same percentage of free amino groups, about 50%,, and roughly the same biological activity, close to 50% of that of the original insulin activity. The statement of Fraenkel-Conrat (l), that a large decrease in the percentage of free groups causes but little change in the biological activity, could also be verified in case of the thiocarbamyl insulin derivatives. This was demonstrated by repeating the isothiocyanate treatment of insulin several times and comparing the free amino groups and biological activities at each step. A progressive decrease in free amino groups
THIOCARBAMYL
DERIVATIVES
OF
97
INSULIN
occurred while the biological activity decreased to 50% with the first treatment and was not substantially reduced with further treatment. These results are shown in Table III. Thus other factors, besides the free amino groups, seem to influence the biological activity. The terminal amino groups involved in the isothiocyanate reaction were determined by a modification of the Edman procedure (2, 3). The thiocarbamyl insulins were hydrolyzed with 2 N hydrochloric acid, and the thiohydantoins were extracted with ether. Instead of hydrolyzing the thiohydantoins to amino acids, as was done by Edman, they were separated by paper chromatography. The location of the various thiohydantoins on the chromatogram could be detected by spraying with TABLE III E$ect of Repetition of the Ally1 Isothiocyanate Reaction on the Free Amino of Allylthiocarbamyl Insulin Derivatives and Biological Activity Crystalline insulin lot
Number of reactions
3 4 3 4 3 4
1 1 2 2 3 3
Total reaction time
Free NHn groups
hr.
Lie
6 6 12 12 18 18
60 70 41 44 26 24
Groups
Biological activity YO
68 f 64 f 67 i 53~ 45* 49zk
10 10 10 8 7 7
Grote’s reagent, a solution containing sodium nitroprusside (5). While the thiohydantoins of most of the amino acids showed blue colors with this reagent, the glycine and phenylalanine derivatives were, respectively, red and yellow. These distinctive colors were exhibited by the thiohydantoins from the insulin samples. In addition, the Rf values of the end-group derivatives were determined in two solvent systems and found to correspond to those of the glycine and phenylalanine reference compounds. No other thiohydantoins were detected. These results are in agreement with the findings of Sanger (6) for the terminal amino acids of the insulin molecule. EXPERIMENTAL
Assay Procedures The free amino groups were determined according to the Moore and Stein method (7). In different batches of crystalline insulin the NH2 equivalents/l04 g. insulin varied from 4.05 to 4.20. The free amino groups of the thiocarbamyl insulin
98
KAISER,
MAXWELL,
LASDMAKN
AND
HUBATA
derivatives were expressed as the percentage of the amino groups of the crystalline insulin from which they were prepared.
Insulin
Activity
Potency was assayed by the mouse convulsion method. In each determination 100 mice were used for the insulin standard and 100 mice for the thiocarbamyl derivatives. The activity of different batches of crystalline insulin was 24-27 units/mg.
Preparation
of Phenylthiocarbamyl
Insulin
in Pyridine-Waler
Mixture
Crystalline zinc insulin was reacted with phenyl isothiocyanate according to t.he method of Edman (2, 3). The thiocarbamyl insulin was precipitated from the reaction mixture at pH 5.5. This preparation had 19% of the original amino groups free and was found to be inactive by the mouse convulsion assay.
Preparation
of Phenylthiocarbamyl Derivatives of Insulin Phosphate Bufer Solutions
in
In preparing these derivatives three different weight ratios between insulin and phenylisothiocyanate were used: 1 part of insulin to 10 parts of phenyl isothiocyanate (10’ g./710 moles); 1 part of insulin to 1 part of phenyl isothiocyanate (10’ g./71 moles); and 5 parts of insulin to 1 part of phenyl isothiocyanate (10’ g./14 moles). The reaction was carried out in a phosphate buffer solution, pH 7.67, which contained 0.064 mole KOH and 0.072 mole KHtPO,/I. Concentration of the insulin in the solution was 1%. With some crystalline insulin preparations the solution was not clear but contained a small amount of suspended matter. Phenyl isothiocyanate was added to the well-stirred insulin solution and the stirring was continued for 2, 6 and 24 hr., respectively. The temperature was kept at 15-20”. The mixture was then transferred into centrifuge tubes and spun until a clear aqueous upper layer separated. This was removed and the lower layer, which consisted of unreacted phenyl isothiocyanate and of a solid, was extracted repeatedly with ether. The ether-insoluble residue was suspended in water, freezedried, and then dried in a vacuum desiccator. Yields of phenylthiocarbamyl insulin precipitates, their free amino groups and biological activity are listed in Table I. These preparations were compared for blood sugar-lowering activity with SPH insulin in fasted rabbits. The total mean blood sugars of five individual assay runs of thiocarbamyl insulin precipitates did not vary significantly from that of NPH insulin at 3-, 5-, and 7-hr. periods. The blood sugar curves were practically superimposed on each other. In a modification of the above procedure the insulin-phenyl isothiocyanate reaction mixture wss not separat.ed by centrifugation but was shaken with ether. The precipitate almost completely dissolved in t,he aqueous layer, which was separated from the ether layer and dialyzed at 4” against distilled water. The salt-free solution was freeze-dried, and the residue was dried in a vacuum desiccator. The phenylthiocarbamyl insulin derivatives were water-soluble.
THIOCARBAMYL
DERIVATIVES
OF
INSULIN
99
It was also found that the extraction with ether could be avoided. The mixture with the unreacted phenyl isothiocyanate was dialyzed, freeze-dried, and the solid residue washed with ether. This method was more convenient, since the extraction of the buffer solution with ether often resulted in strong emulsification.
Analyses 1. Crystalline Zinc insulin. N, 14.72%; P, 0.02%; and NH,, 4.19 equiv./104 g. 2. Phenylthiocurbamyl Insulin (prepared by weight ratio: 1 part insulin to 10
parts phenylisothiocyanate). 3. Phenylthiocurbamyl
h’, 14.44%; P, 0.11%. (prepared by weight ratio: 5 parts insulin to N, 14.55%; P, 0.09%.
Insulin
1 part phenylisothiocyanate).
Preparation of Allythiocarbamyl
Insulin
Derivatives
Two grams of crystalline insulin was added to 200 ml. of t,he 0.072 M phosphate buffer solut.ion, pH 7.67. To the well-stirred solution, 20 g. of ally1 isothiocysnate (approx. 990 moles/104 g. insulin) was added. Samples of 25 ml. were withdrawn at 1,2,3, and 6 hr. The remaining 100 ml. of the mixture was st,irred for a total of 24 hr. In none of the fractions did precipitation occur. Each fraction was processed in an identical manner. The unreacted ally1 isothiocyanate was extracted with ether, and the aqueous layer was brought to pH 5.5. The precipitate formed at this pH was removed by centrifugation and the solid was suspended in distilled water and freeze-dried. A total of 1.9 g. of allylisothiocarbamyl insulin was recovered from the fractions. Analytical and bioassay results are listed in Table II.
Repetition of the Ally1 Isothiocyanate Reaction One part of crystalline insulin and 10 parts of ally1 isothiocyanate were reacted for 6 hr. as described above. Part of the product was used for the determination of the free amino groups and for assaying the insulin activity by the mouse convulsion method. The residue of the material was treated again with ally1 isothiocyanate for another 6-hr. period. The whole process was repeated for a third time. Bioassay results by the mouse convulsion test and free amino group determinations are given in Table III.
Color Reaction for Thiohydantoins The reagent described by Grote (5) was slightly modified. The stock solution was prepared as directed and allowed to stand for 24 hr. Bfter all the insoluble material, which appeared on standing, had been removed by filtration, the cleal solution was diluted to the specified volume and stored in a dark bottle. Immediately before use, this solution was diluted with an equal volume of water, saturated with sodium bicarbonate, and filtered. The resulting solution was applied to the paper as a spray. Although the colors usually developed within a short time, maximum intensity could be achieved more quickly and satisfactorily by exposing the moist paper to an atmosphere of steam for a few minutes.
100
KAISER,
MAXWELL,
LANDYAh-N
AND
HUBATA
Hydrolysis of Thiocarbam yl Insulins After several preliminary trials the following hydrolysis procedure was adopted: Ten milligrams of a thiocarbamyl insulin sample was placed in a small test tube, and 0.5 ml. of 2 N hydrochloric acid was added. The tube was immersed in a boiling water bath for 1 hr. After cooling, the mixture was repeatedly ext.racted with ether, and the ether was dried and concentrated. Part of the concentrated ether solution was used for the identification of the thiohydantoins by paper chromatography.
Paper Chromatography of the Thiohydantoins One drop of the concentrated et.her solution of t.he thiohydantoins was placed on Whatman ~4 filter paper which was previously impregnated with a 0.05 M phthalate buffer at pH 6 (8). Secondary butanol, saturated with the buffer, was the running solvent. Comparison of the spots obtained with the corresponding phenylalanine and glycine thiohydantoin reference compounds, which were run simultaneously, revealed phenylalanine and glycine as the terminal amino acids of insulin. So other thiohydantoins were detected when the ether extracts of the thiocarbamyl insulin hydrolyzates were chromatographed in a second system, xyleneacetic acid-pH 6 buffer (3:2:1). In this system, the glycine and phenylalanine derivatives have widely different RI values. This, together with their distinctive colors in Grate’s solution, enables them to be dietinguished unequivocally from the other amino acid thiohydantoins. SThese results were further confirmed by hydrolysis of the thiohydantoins with barium hydroxide (2, 3) to the free amino acids, which, when chromatographed by the usual techniques, proved to be phenylalanine and glycine. ACKNOWLEDGMENTS
The authors wish to thank Mr. B. W. Petty and Mr. R. W. Scott and their coworkers for the insulin aSBay reported in this paper. &JMMARY
Insulin was reacted with phenyl isothiocyanate and with ally1 isothiocyanate. When this reaction was carried out in aqueous buffer solutions, biologically active phenylthiocarbamyl and allylthiocarbamyl insulin derivatives were obtained. Solubility properties, biological activity, and the amount of free amino groups were determined for a number of these insulin derivatives. A color reaction was applied to the identification of tbiohydantoins. This reaction was also used in the paper chromatography of allyl- and phenylthiocarbamyl insulin hydrolylzates. The terminal amino acids 8 A more detailed discussion of the chromatography of amino acid thiohydantoins with a list of RI values will appear in a subsequent paper, now in press, by W. A. Landmann, J. Dillaha, and 11. Drake.
TIIIOCARBAMYL
DERIVATIVES
OF
101
INSI‘LIN
involved in the reaction with the isothiocyanates were identified by their thiohydantoins in the chromatograms of the ether extracts of the of the insulin derivatives. Complete agreement with hydrolyzates Sanger’s findings is reported. REFERENCES I. FRAESKEL-CONRAT,
J., AND FRAENKEL-COSHAT,
H.,
Biochim.
et Biophys.
Acfu 6, 89 (1950). 2. ~CDMAN, I’., Ada Chem. Stand. 4, 277 (1950). 3. EDMAS, P., Acta Chem. Scud. 4, 28.3 (1950). 4. FRAESKEL-COXRAT, H., AND FRAENKEL-CONRAT, (1951). 5. GROTE, I. W., J. Biol. Chem. 93, 25 (1931).
J., Acta Chem.Scund.6,409
6. SAXGER, b’., Biochem. J. 39, 507 (1945). 7. MOORE:, S., ANI) STEIN, W., J. Biol. Chem. 176, 367 (1948). 8. HAWK, 1’. l%., OSER, B. L., AND SUM%~ERSON, W. H., I’raetictlI
Chemist,ry, 12th Ed., p 26. The Mlrkiston Co., Philndelphia,
Physiological 1947.
Active Thiocarbamyl
Derivatives
of Insulins
Emil Kaiser,2 L. C. Maxwell, W. A. Landmann and Robert Hubata From the Armour
Laboratories,
Chicago,
Illinois
Received August 18, 1952
The functional groups responsible for the biological activity of insulin have been studied by a number of investigators. A detailed discussion of this subject was recently published by J. and H. Fraenkel-Conrat (1). According to these authors, several groups, among them the free amino groups, may be largely involved in chemical reactions without any apparent loss of insulin activity. To broaden t,he study of the correlation between insulin activity and the number of free amino groups in the insulin molecule, the isothiocyanate reaction of Edman (2, 3) was applied to insulin. The use of the Edman procedure for the determination of the amino acid sequence in insulin was reported recently (4), but the biological activity of the t.hiocarbamyl derivatives was not studied. The react,ion between peptides and phenyl isothiocyanate was carried out by Edman in pyridinewater mixt,ures. Application of this procedure to insulin yielded biologically inactive products in our hands. Therefore it was attempted to use aqueous buffer solutions instead of pyridinewater mixtures. The insulin derivatives prepared in buffer solutions were biologically active when tested by the mouse convulsion assay. RESULTS
When phenyl isothiocyanate was added to a solution of insulin in a phosphate buffer at pH 7.67, a precipitate was slowly formed. The amount of the precipitated insulin depended upon the reaction time, and 1 Presented before the Division of Biological Chemistry, American Chemical Society, Milwaukee, March 31, 1952. * Present address: The Bio-l’rocess Co., Division of Armour h Co., .Joliet, 111. 94
THIOCARBAMYL
DERIVATIVES
OF
95
INSULIN
upon the weight ratio between insulin and phenyl isothiocyanate (Table I). When 10 parts of phenyl isothiocyanate per 1 part of insulin was used, corresponding to 710 moles of phenyl isothiocyanate/104 g. insulin, a reaction time of 24 hr. was needed to precipitate the insulin completely at temperatures of 15-20”. Seventy-one moles of phenyl isothiocyanate/104 g. insuli21, 1 part of phenyl isothiocyanat)e per 1 part of insulin, yielded only 60% of insulin precipitate within 24 hr. In these produck about 4(t-50y0 of the amino groups remained unreacted. The TABLE Free Amino
Phenyl isothiocyam&/l0’ g. insulin
710 710 710 71 71 71 14 0
Nitrogen
Reaction
time
I
and Biological Activity Insulin Derivatives Recovery of insulin in ppt.”
of Phenylthiocnrbamyl
Free NHz
groupsb
hr.
%
%
2 6 24 2 6 24 24 0
22 44 90 25 40 53 35
54 44 44 49 38 41 65 100
Biological
activityC
5
57 48 52 54 44 47 80 100
f f z!z f f f f f
8 7
8 8 7 7 12 10
a Averages of three determinations. b Averages of three duplicate determinations. c Biological activity was measured in the mouse convulsion Ii. S. P. reference standard.
test against
the
insulin activity, determined according to the mouse convulsion test, was about 50% of the original activity. When 14 moles of phenyl isothiocyanate were used for lo4 g. insulin, 1 part of phenyl isothiocyanate per 5 parts of insulin, the yield of the precipitate amounted to 35% only. This preparation had 65y0 of the free amino groups unchanged and about 80% of the original activity was retained. All of the phenylthiocarbamyl insulin precipitates showed prolonged blood sugar-lowering activity, similar to that of the neutralprotamine-of-Hagedorn (NPH) insulin, when tested in fasted rabbits. The phenylthiocarbamyl insulin precipitates were isolated from the buffer solutions by centrifugation in the presence of Ohe unreacted phenyl isothiocyanate. When the phenyl isothiocyanate was extracted
96
KAISER,
MAXWELL,
LANDMANN
AND
HUBATA
with ether before centrifugation, the precipitate dissolved in the buffer solution. The phenylthiocarbamyl insulin could be separated from the buffer by dialysis and subsequent freeze-drying of the dialyzed solution. These products were water-soluble, yielding a solution of pH 6.9-7.0. The dependence of the formation of an insulin precipitate at pH 7.67 upon the buffer-phenyl isothiocyanate system was further demonstrated when ally1 isothiocyanate was used instead of phenyl isothiocyanate. Ten parts of ally1 isothiocyanate per 1 part of insulin, 990 moles of ally1 isothiocyanate/104 g. insulin, did not precipitate an insulin
The Influence Biological Reaction
time
TABLE II of Reaction Time on the Free Amino GTOUPS and the Activity of Allylthiocarbamyl Insulin Derivatives Insulin
recovered as ppt.”
hr.
0
1 2 3 6 24
% -
85-90 85-90 85-90 85-90 85-90
Free NH,
group8
Biological
% 100
73 75 71 57 50
activityC %
loo
f
83 f 70 f 84 f 68 f 55f
10 12 10 13 10 8
DAverages of three determinations. b Averages of three duplicate determinations. c Biological activity was measured in the mouse convulsion test against the U. S. P. reference standard.
derivative from a phosphate buffer solution at pH 7.67. The allylthiocarbamyl insulin precipitated when the pH was adjusted to 5.5. The initial reaction rate of insulin with ally1 isothiocyanate was somewhat slower than with phenyl isothiocyanate (Tables I and II). But after 24 hr. both the ally1 and the phenyl products had approximately the same percentage of free amino groups, about 50%,, and roughly the same biological activity, close to 50% of that of the original insulin activity. The statement of Fraenkel-Conrat (l), that a large decrease in the percentage of free groups causes but little change in the biological activity, could also be verified in case of the thiocarbamyl insulin derivatives. This was demonstrated by repeating the isothiocyanate treatment of insulin several times and comparing the free amino groups and biological activities at each step. A progressive decrease in free amino groups
THIOCARBAMYL
DERIVATIVES
OF
97
INSULIN
occurred while the biological activity decreased to 50% with the first treatment and was not substantially reduced with further treatment. These results are shown in Table III. Thus other factors, besides the free amino groups, seem to influence the biological activity. The terminal amino groups involved in the isothiocyanate reaction were determined by a modification of the Edman procedure (2, 3). The thiocarbamyl insulins were hydrolyzed with 2 N hydrochloric acid, and the thiohydantoins were extracted with ether. Instead of hydrolyzing the thiohydantoins to amino acids, as was done by Edman, they were separated by paper chromatography. The location of the various thiohydantoins on the chromatogram could be detected by spraying with TABLE III E$ect of Repetition of the Ally1 Isothiocyanate Reaction on the Free Amino of Allylthiocarbamyl Insulin Derivatives and Biological Activity Crystalline insulin lot
Number of reactions
3 4 3 4 3 4
1 1 2 2 3 3
Total reaction time
Free NHn groups
hr.
Lie
6 6 12 12 18 18
60 70 41 44 26 24
Groups
Biological activity YO
68 f 64 f 67 i 53~ 45* 49zk
10 10 10 8 7 7
Grote’s reagent, a solution containing sodium nitroprusside (5). While the thiohydantoins of most of the amino acids showed blue colors with this reagent, the glycine and phenylalanine derivatives were, respectively, red and yellow. These distinctive colors were exhibited by the thiohydantoins from the insulin samples. In addition, the Rf values of the end-group derivatives were determined in two solvent systems and found to correspond to those of the glycine and phenylalanine reference compounds. No other thiohydantoins were detected. These results are in agreement with the findings of Sanger (6) for the terminal amino acids of the insulin molecule. EXPERIMENTAL
Assay Procedures The free amino groups were determined according to the Moore and Stein method (7). In different batches of crystalline insulin the NH2 equivalents/l04 g. insulin varied from 4.05 to 4.20. The free amino groups of the thiocarbamyl insulin
98
KAISER,
MAXWELL,
LASDMAKN
AND
HUBATA
derivatives were expressed as the percentage of the amino groups of the crystalline insulin from which they were prepared.
Insulin
Activity
Potency was assayed by the mouse convulsion method. In each determination 100 mice were used for the insulin standard and 100 mice for the thiocarbamyl derivatives. The activity of different batches of crystalline insulin was 24-27 units/mg.
Preparation
of Phenylthiocarbamyl
Insulin
in Pyridine-Waler
Mixture
Crystalline zinc insulin was reacted with phenyl isothiocyanate according to t.he method of Edman (2, 3). The thiocarbamyl insulin was precipitated from the reaction mixture at pH 5.5. This preparation had 19% of the original amino groups free and was found to be inactive by the mouse convulsion assay.
Preparation
of Phenylthiocarbamyl Derivatives of Insulin Phosphate Bufer Solutions
in
In preparing these derivatives three different weight ratios between insulin and phenylisothiocyanate were used: 1 part of insulin to 10 parts of phenyl isothiocyanate (10’ g./710 moles); 1 part of insulin to 1 part of phenyl isothiocyanate (10’ g./71 moles); and 5 parts of insulin to 1 part of phenyl isothiocyanate (10’ g./14 moles). The reaction was carried out in a phosphate buffer solution, pH 7.67, which contained 0.064 mole KOH and 0.072 mole KHtPO,/I. Concentration of the insulin in the solution was 1%. With some crystalline insulin preparations the solution was not clear but contained a small amount of suspended matter. Phenyl isothiocyanate was added to the well-stirred insulin solution and the stirring was continued for 2, 6 and 24 hr., respectively. The temperature was kept at 15-20”. The mixture was then transferred into centrifuge tubes and spun until a clear aqueous upper layer separated. This was removed and the lower layer, which consisted of unreacted phenyl isothiocyanate and of a solid, was extracted repeatedly with ether. The ether-insoluble residue was suspended in water, freezedried, and then dried in a vacuum desiccator. Yields of phenylthiocarbamyl insulin precipitates, their free amino groups and biological activity are listed in Table I. These preparations were compared for blood sugar-lowering activity with SPH insulin in fasted rabbits. The total mean blood sugars of five individual assay runs of thiocarbamyl insulin precipitates did not vary significantly from that of NPH insulin at 3-, 5-, and 7-hr. periods. The blood sugar curves were practically superimposed on each other. In a modification of the above procedure the insulin-phenyl isothiocyanate reaction mixture wss not separat.ed by centrifugation but was shaken with ether. The precipitate almost completely dissolved in t,he aqueous layer, which was separated from the ether layer and dialyzed at 4” against distilled water. The salt-free solution was freeze-dried, and the residue was dried in a vacuum desiccator. The phenylthiocarbamyl insulin derivatives were water-soluble.
THIOCARBAMYL
DERIVATIVES
OF
INSULIN
99
It was also found that the extraction with ether could be avoided. The mixture with the unreacted phenyl isothiocyanate was dialyzed, freeze-dried, and the solid residue washed with ether. This method was more convenient, since the extraction of the buffer solution with ether often resulted in strong emulsification.
Analyses 1. Crystalline Zinc insulin. N, 14.72%; P, 0.02%; and NH,, 4.19 equiv./104 g. 2. Phenylthiocurbamyl Insulin (prepared by weight ratio: 1 part insulin to 10
parts phenylisothiocyanate). 3. Phenylthiocurbamyl
h’, 14.44%; P, 0.11%. (prepared by weight ratio: 5 parts insulin to N, 14.55%; P, 0.09%.
Insulin
1 part phenylisothiocyanate).
Preparation of Allythiocarbamyl
Insulin
Derivatives
Two grams of crystalline insulin was added to 200 ml. of t,he 0.072 M phosphate buffer solut.ion, pH 7.67. To the well-stirred solution, 20 g. of ally1 isothiocysnate (approx. 990 moles/104 g. insulin) was added. Samples of 25 ml. were withdrawn at 1,2,3, and 6 hr. The remaining 100 ml. of the mixture was st,irred for a total of 24 hr. In none of the fractions did precipitation occur. Each fraction was processed in an identical manner. The unreacted ally1 isothiocyanate was extracted with ether, and the aqueous layer was brought to pH 5.5. The precipitate formed at this pH was removed by centrifugation and the solid was suspended in distilled water and freeze-dried. A total of 1.9 g. of allylisothiocarbamyl insulin was recovered from the fractions. Analytical and bioassay results are listed in Table II.
Repetition of the Ally1 Isothiocyanate Reaction One part of crystalline insulin and 10 parts of ally1 isothiocyanate were reacted for 6 hr. as described above. Part of the product was used for the determination of the free amino groups and for assaying the insulin activity by the mouse convulsion method. The residue of the material was treated again with ally1 isothiocyanate for another 6-hr. period. The whole process was repeated for a third time. Bioassay results by the mouse convulsion test and free amino group determinations are given in Table III.
Color Reaction for Thiohydantoins The reagent described by Grote (5) was slightly modified. The stock solution was prepared as directed and allowed to stand for 24 hr. Bfter all the insoluble material, which appeared on standing, had been removed by filtration, the cleal solution was diluted to the specified volume and stored in a dark bottle. Immediately before use, this solution was diluted with an equal volume of water, saturated with sodium bicarbonate, and filtered. The resulting solution was applied to the paper as a spray. Although the colors usually developed within a short time, maximum intensity could be achieved more quickly and satisfactorily by exposing the moist paper to an atmosphere of steam for a few minutes.
100
KAISER,
MAXWELL,
LANDYAh-N
AND
HUBATA
Hydrolysis of Thiocarbam yl Insulins After several preliminary trials the following hydrolysis procedure was adopted: Ten milligrams of a thiocarbamyl insulin sample was placed in a small test tube, and 0.5 ml. of 2 N hydrochloric acid was added. The tube was immersed in a boiling water bath for 1 hr. After cooling, the mixture was repeatedly ext.racted with ether, and the ether was dried and concentrated. Part of the concentrated ether solution was used for the identification of the thiohydantoins by paper chromatography.
Paper Chromatography of the Thiohydantoins One drop of the concentrated et.her solution of t.he thiohydantoins was placed on Whatman ~4 filter paper which was previously impregnated with a 0.05 M phthalate buffer at pH 6 (8). Secondary butanol, saturated with the buffer, was the running solvent. Comparison of the spots obtained with the corresponding phenylalanine and glycine thiohydantoin reference compounds, which were run simultaneously, revealed phenylalanine and glycine as the terminal amino acids of insulin. So other thiohydantoins were detected when the ether extracts of the thiocarbamyl insulin hydrolyzates were chromatographed in a second system, xyleneacetic acid-pH 6 buffer (3:2:1). In this system, the glycine and phenylalanine derivatives have widely different RI values. This, together with their distinctive colors in Grate’s solution, enables them to be dietinguished unequivocally from the other amino acid thiohydantoins. SThese results were further confirmed by hydrolysis of the thiohydantoins with barium hydroxide (2, 3) to the free amino acids, which, when chromatographed by the usual techniques, proved to be phenylalanine and glycine. ACKNOWLEDGMENTS
The authors wish to thank Mr. B. W. Petty and Mr. R. W. Scott and their coworkers for the insulin aSBay reported in this paper. &JMMARY
Insulin was reacted with phenyl isothiocyanate and with ally1 isothiocyanate. When this reaction was carried out in aqueous buffer solutions, biologically active phenylthiocarbamyl and allylthiocarbamyl insulin derivatives were obtained. Solubility properties, biological activity, and the amount of free amino groups were determined for a number of these insulin derivatives. A color reaction was applied to the identification of tbiohydantoins. This reaction was also used in the paper chromatography of allyl- and phenylthiocarbamyl insulin hydrolylzates. The terminal amino acids 8 A more detailed discussion of the chromatography of amino acid thiohydantoins with a list of RI values will appear in a subsequent paper, now in press, by W. A. Landmann, J. Dillaha, and 11. Drake.
TIIIOCARBAMYL
DERIVATIVES
OF
101
INSI‘LIN
involved in the reaction with the isothiocyanates were identified by their thiohydantoins in the chromatograms of the ether extracts of the of the insulin derivatives. Complete agreement with hydrolyzates Sanger’s findings is reported. REFERENCES I. FRAESKEL-CONRAT,
J., AND FRAENKEL-COSHAT,
H.,
Biochim.
et Biophys.
Acfu 6, 89 (1950). 2. ~CDMAN, I’., Ada Chem. Stand. 4, 277 (1950). 3. EDMAS, P., Acta Chem. Scud. 4, 28.3 (1950). 4. FRAESKEL-COXRAT, H., AND FRAENKEL-CONRAT, (1951). 5. GROTE, I. W., J. Biol. Chem. 93, 25 (1931).
J., Acta Chem.Scund.6,409
6. SAXGER, b’., Biochem. J. 39, 507 (1945). 7. MOORE:, S., ANI) STEIN, W., J. Biol. Chem. 176, 367 (1948). 8. HAWK, 1’. l%., OSER, B. L., AND SUM%~ERSON, W. H., I’raetictlI
Chemist,ry, 12th Ed., p 26. The Mlrkiston Co., Philndelphia,
Physiological 1947.