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Investigations on proteins and polymers. VI. Interaction of egg albumin with a thiophene-2-azlactone
Investigations on Proteins and Polymers. VI, ,kteraction of Egg Albumin with a Thiophene-Z-Azlactone Serge N. Timasheff and F. F. Nord From the Department of Organic Chemistry and Enzynwlogy,t * Far&urn Univera& Nao Y&c 68, New York Received November 30, 1950
In studies on the influence of various groups upon the biological and physicochemical properties (1) of proteins, the blocking of these groups by various chemical means has received considerable attention (2). The interaction between a protein and various azlactones has been reported by Lettrd and co-workers (3). These investigators have shown by means of anaphylactic tests that the material obtained differed from the original product. However, no proof of the formation of a chemical compound was offered. The neprly developed synthesis of thiophene azlactones (4) starting from thiophene-2-aldehyde (5) has made a number of these compounds available. Due to the reported biological activity of thienyl alanine (6) and the resolution thereof into its two optical isomers (7), it became of interest to attempt the introduction of similar groups into a protein molecule. Therefore, egg albumin was caused to interact with a thiophene-2-azlactone, namely, 2-phenyl-4-(Zthenal)-5oxazolone, with the formation of a chemically modified acidic protein. EXPERIMENTAL
Preparation of Modified Protein The preparation of the egg albumin was described in the previous paper of this series (8). To 50 ml. of a 3yo solution of egg albumin there were added the calculated 1 Communication No. 212. See aleo preceding paper in this series. This study wae carried out under the auspices of the Atomio Energy Commission. Presented before the Division of Biological Chemistry of the American Chemical Society, Chicago, Ill., September, 1950. * The data recorded have been condensed from a part of the dksertation of S. N. T. eubmitted to the Graduate School of Fordham University in partial fulfillment of the requirements for the Ph.D. degree, 1951. 320
VI.
EGG
ALBUMIN
AND
321
THIOPHENl!J+!-AZLACTONE
amount of 2-phenykk(2-thenal)&oxazolone dissolved in 30 ml. dioxane and the amount of 1 N NaOH equivalent to the oxazolone. The mixture was allowed to stand at room temperature for 48 hr. at pH 8.0, at the end of which period it was neutralized with 1 N HCl. Upon addition of the acid, a yellowish gelatinous precipitate formed. This precipitate was purified five times by dissolving it in base and reprecipitating it in acid. The material was then dialyzed against running distilled water for 48 hr., dilute KC1 for 24 hr. to precipitate the modified protein, and again against distilled water for 48 hr. The precipitated material was then dissolved and stored at pH 7.6.
Preparation
oj Model Compounds
The a-benzamido-fi-2-thienylacrylic acid and its methyl and ethyl esters were prepared according to previously described methods (9). The n-octylamide of the acrylic acid was obtained according to the method suggested (10) for similar compounds in the benzene series. To 0.5 g. of n-octylamine dissolved in 20 ml. of alcohol there was added 1 g. of 2-phenyl-4-(2-thenal)5-oxazolone. The reaction mixture was heated then on a steam bath under refiux, the azlactone dissolving after 20 min. After 2 hr. of reflux the solution was allowed
./-\ ,~,..--.. //. /I.’
. “.>
60 .:A
~_. . . . _. . . . . . 200
t
I
215
\ ::
\
\ ‘\ 245
.A.-.-.-./.C.275-.-.--x .--. 305 I
I
335
Wave length, rn/! FIG. 1. Ultraviolet absorption spectra. Left scale: - o o - cr-benzamido+2-thienylacrylic acid, - - - n-octylamide of ,%Rhienylacrylic acid, -.-.-.methyl ester of ,P2&ienylacrylic acid, ....... . ethyl ester of &2-thienylacrylic acid, xxxxxxx 2-phenyl-4-(2-thenal)+oxazolone. Right scale: -.-.egg albumin, egg albumin derivative of a-benzamido-&2-thienylacrylic acid,
322
8. N. TIbfASHEFF
AND F. F. NORD
to cool and a little water was added, a yellow oil separating out. After 2 days of standing at - lO”C., the oil had solidified. It, was then filtered off and recrystallized from alcohol. Yield: quantitative. Analysis: Calcd. for C~~HZSOINZS:C 08.93, H 7.05; found: C 68.60, H 6.95.
Measurements The ultraviolet absorption spectra were determined on a Beckman quartz model DU spectrophotometer. The free primary amino groups were determined as usual (11). The electrophoretic analyses were carried out, on a Perkin-Elmer Tiselius electrophoresis apparatus, model 38 (12). The mobilities were calculated using the procedure of Longsworth and McInnes (13). RESULTS
AND DISCUSSION
The ultraviolet absorption spectrum of the new product and those of egg albumin, the pure azlactone, the free acrylic acid and various derivatives of it are presented in Fig. 1. The curve of the modified protein displays two maxima, one at 275-280 ml.c, characteristic for pure egg albumin and the acrylic acid and its derivatives, the other at 310-315 ml.c, typical
for the acrylic acid and its derivatives.
However,
the absorption maxima at 270 ml.cand 393-395 rnp, characteristic for the azlactone (4) are absent in the reaction product. From these ultraviolet
data it can be concluded that the
absorption
obtained material is a compound formed between egg albumin and the azlactone via the opening of its ring as shown in the following:
\
I -
I
Egg albumin
-
b =o L X = NH, 0, 5, = N
& 6%
,Sl
VI.
EGG ALBUMIN
AND
323
THIOPHENE-%-AZLACTONE
The substituted protein is acidic in nature precipitating below pH 7 but remaining in solution at higher values of pH, indicating that the main mode of interaction is via the blocking of the amino groups of the protein. To determine whether linkages of other types between the thienylacrylic acid residues and the protein are formed, a study was carried out on samples of the protein with various degrees of substitution. The total thienylacrylic acid substitution in each sample was estimated by means of the ultraviolet absorption intensity in the regions of 275-280 rnp and 310-315 mp.3 The reaction with amino groups could TABLE Analytical
Sample no.
= I
Van Slyke data
-
N&m&in
_-
-mp./g. 0.0 0.0
1 2 3 4 5 6 7 8”
I Compound
Data on Egg Albumin-Thiophene&ada&me
No. of amino i:roups blockec 1
19 15 14 13.5 11 6.5 0
-
-
-
data IZ:7m,. 310-15 mp
275-80 mp
Electrophoretic mobilitiea x 10’
-24.8 19.6 13.5 9.3 8.0 5.3 3.2 0.2
23.1 20.6 17.0 13.7 13.3 11.6 9.6 7.1
19
1.6 1.8 2.1 3.0 4.7 7.2
-
ZXZ Ultraviolet _-I
cm./sec./a./cm. - 1.0
-0.99 -0.92
-0.81 -0.81 -0.79 -0.73 -0.71
-
a Pure egg albumin.
be followed by Van Slyke analysis of residual free amino groups. The electrophoretic analyses indicated the degree of change in the electrokinetic properties of the protein. The ultraviolet data are recorded in ~01s.4 and 5 of Table I. The Van a Sulfur analyses were carried out on all the samples. Although a definite gradation in sulfur content was found with increasing substitution, no quantitative conclusion could be reached, the values being low in all cases. When samples of egg albumin were subjected to the exact conditions of the azlactone introduction into it, it was found that their sulfur content had decreased. Thus, while native recrystallized egg albumin gave a sulfur content of 1.54yo, the treated control samples contained only 0.85-1.22~o. Such loss of sulfur atoms during this treatment at pH 8.0 and room temperature explains adequately the lack of agreement in the quantitative gradation of the sulfur values.
324
8. N. TIMASHEFF
AND
F. F. NORD
Slyke free amino group determinations and the electrophoretic mobilities are presented in ~01s.2, 3, and 6. The electrophoretic patterns -are shown in Fig. 2. From these data it can be seen that the total number of thienylacrylic acid residues introduced into the protein is not accounted for by the number of blocked amino groups for each particular sample, as the E:&. values increase after all the amino groups had reacted (samples
s&A 5,400 3,600 sec. sec. +DescendingFro. 2. Electrophoretic
A” 3,600 5,400 sec. sec. --Ascending-,
patterns of egg albumin-thiophene2azlactone PH = 7.5 (0.1 M phosphate buffer).
compound.
1 and 2). Therefore, it must be concluded that other types of interaction also occur, possibly with the serine or tyrosine hydroxyl, cysteine sulfhydryl, or proline secondary amino groups, all of which are known to be able to react with.azlactones. Furthermore, from data on samples 3, 4, and 5 it can be seen that while the total number of acrylic acid residues entering into the protein molecule is constantly increasing, the number of amino groups readily reacting seems to be limited to 70-75% of the total amino content. Complete saturation, however, of all amino groups takes place only in
VI.
EQG ALBUMIN
AND
THIOPHENE-2-AZLACTONE
325
cases of a large excess of adactone in the reaction mixture (samples 1 and 2). This could point to the presence of two types of amino groups in egg albumin, radically differing in the degree of reactivity, a fact which could also be deduced from results previously reported in the literature (14). The observed heterogeneity in the reactivity of amino groups of egg albumin could also be attributed to a possible difference in reactivity of the various fractions (15) found to be present in that protein. SUMMARY
A new modified protein was prepared by the reaction between egg albumin and a thiophene-2-azlactone. This acid polymer was shown by an ultraviolet absorption study to be a compound formed of egg albumin and a thienylacrylic acid. In this reaction, the principal reactive groups are the primary amino groups of the protein. However, other groups are also entering into competition, as only 70-75% of the amino groups were found to be strongly reactive. REFERENCES 1. BIER, M., AND Nom, F. F., Proc. Natl. Acad. Sci. U. S. 36, 17 (1949). 2. HERRIOTT, R. M., Advances in Protein Chem. 3, 170 (1947). H., AND 3. LEW&, H,, AND HAAG, R., 2. physiol. Chem. 266, 31 (1940); LEE, FERNHOLZ, M. E., ibid. 206, 37 (1940); LEYIYU%, H., BUCHHOLZ, K., AND FERNEOLZ, M. E., ibid. 267, 108 (1940). 4. CROWE, B. F., AND NORD, F. F., J. Org. Chem. 16, 81 (1950). 5. KING, W. J., AND Nom, F. F., J. Org. Chem. 13, 635 (1948). 6. FERGIER,M. F., AND VIQNEAUD, V. DU, J. Biol. Chem. 179, 61 (1949). 7. CROWE,B. F., AND Nom, F. F., J. Org. Chem. 15, 688 (1950). 8. TIMASAEFF, S. N., AND NORD, F. F., Arch. Biochem. Biophys. 31, 309 (1951). 9. CROWE,B. F., AND Nom, F. F., J. Org. Chem. 16, 1177 (1950). 10. GE&ACHER, C., AND GKJLBAS,G., Helv. Chim. Ada 10, 819 (1927). 11. VAN SLYKE, D. D., J. Biol. Chem. 16, 121 (1913). 12. MOORE, D. H., AND WHITE, J. U., Rev. Sci. ~netrumem% 16, 700 (1948). 13. LONQSWORTH, L. G., AND MACINNES, D. A., J. Am. Chem. Sot. 62,705 (1940). 14. KLECZKOWSIZI, A., &it. J. Ecptl. Path. 21, 1 (1940). 15. CANN, J. R., AND KIRKWOOD, J. G., Cold Spring Harbor Symposia 012Quant. Biol. 14, 9 (1950).
In studies on the influence of various groups upon the biological and physicochemical properties (1) of proteins, the blocking of these groups by various chemical means has received considerable attention (2). The interaction between a protein and various azlactones has been reported by Lettrd and co-workers (3). These investigators have shown by means of anaphylactic tests that the material obtained differed from the original product. However, no proof of the formation of a chemical compound was offered. The neprly developed synthesis of thiophene azlactones (4) starting from thiophene-2-aldehyde (5) has made a number of these compounds available. Due to the reported biological activity of thienyl alanine (6) and the resolution thereof into its two optical isomers (7), it became of interest to attempt the introduction of similar groups into a protein molecule. Therefore, egg albumin was caused to interact with a thiophene-2-azlactone, namely, 2-phenyl-4-(Zthenal)-5oxazolone, with the formation of a chemically modified acidic protein. EXPERIMENTAL
Preparation of Modified Protein The preparation of the egg albumin was described in the previous paper of this series (8). To 50 ml. of a 3yo solution of egg albumin there were added the calculated 1 Communication No. 212. See aleo preceding paper in this series. This study wae carried out under the auspices of the Atomio Energy Commission. Presented before the Division of Biological Chemistry of the American Chemical Society, Chicago, Ill., September, 1950. * The data recorded have been condensed from a part of the dksertation of S. N. T. eubmitted to the Graduate School of Fordham University in partial fulfillment of the requirements for the Ph.D. degree, 1951. 320
VI.
EGG
ALBUMIN
AND
321
THIOPHENl!J+!-AZLACTONE
amount of 2-phenykk(2-thenal)&oxazolone dissolved in 30 ml. dioxane and the amount of 1 N NaOH equivalent to the oxazolone. The mixture was allowed to stand at room temperature for 48 hr. at pH 8.0, at the end of which period it was neutralized with 1 N HCl. Upon addition of the acid, a yellowish gelatinous precipitate formed. This precipitate was purified five times by dissolving it in base and reprecipitating it in acid. The material was then dialyzed against running distilled water for 48 hr., dilute KC1 for 24 hr. to precipitate the modified protein, and again against distilled water for 48 hr. The precipitated material was then dissolved and stored at pH 7.6.
Preparation
oj Model Compounds
The a-benzamido-fi-2-thienylacrylic acid and its methyl and ethyl esters were prepared according to previously described methods (9). The n-octylamide of the acrylic acid was obtained according to the method suggested (10) for similar compounds in the benzene series. To 0.5 g. of n-octylamine dissolved in 20 ml. of alcohol there was added 1 g. of 2-phenyl-4-(2-thenal)5-oxazolone. The reaction mixture was heated then on a steam bath under refiux, the azlactone dissolving after 20 min. After 2 hr. of reflux the solution was allowed
./-\ ,~,..--.. //. /I.’
. “.>
60 .:A
~_. . . . _. . . . . . 200
t
I
215
\ ::
\
\ ‘\ 245
.A.-.-.-./.C.275-.-.--x .--. 305 I
I
335
Wave length, rn/! FIG. 1. Ultraviolet absorption spectra. Left scale: - o o - cr-benzamido+2-thienylacrylic acid, - - - n-octylamide of ,%Rhienylacrylic acid, -.-.-.methyl ester of ,P2&ienylacrylic acid, ....... . ethyl ester of &2-thienylacrylic acid, xxxxxxx 2-phenyl-4-(2-thenal)+oxazolone. Right scale: -.-.egg albumin, egg albumin derivative of a-benzamido-&2-thienylacrylic acid,
322
8. N. TIbfASHEFF
AND F. F. NORD
to cool and a little water was added, a yellow oil separating out. After 2 days of standing at - lO”C., the oil had solidified. It, was then filtered off and recrystallized from alcohol. Yield: quantitative. Analysis: Calcd. for C~~HZSOINZS:C 08.93, H 7.05; found: C 68.60, H 6.95.
Measurements The ultraviolet absorption spectra were determined on a Beckman quartz model DU spectrophotometer. The free primary amino groups were determined as usual (11). The electrophoretic analyses were carried out, on a Perkin-Elmer Tiselius electrophoresis apparatus, model 38 (12). The mobilities were calculated using the procedure of Longsworth and McInnes (13). RESULTS
AND DISCUSSION
The ultraviolet absorption spectrum of the new product and those of egg albumin, the pure azlactone, the free acrylic acid and various derivatives of it are presented in Fig. 1. The curve of the modified protein displays two maxima, one at 275-280 ml.c, characteristic for pure egg albumin and the acrylic acid and its derivatives, the other at 310-315 ml.c, typical
for the acrylic acid and its derivatives.
However,
the absorption maxima at 270 ml.cand 393-395 rnp, characteristic for the azlactone (4) are absent in the reaction product. From these ultraviolet
data it can be concluded that the
absorption
obtained material is a compound formed between egg albumin and the azlactone via the opening of its ring as shown in the following:
\
I -
I
Egg albumin
-
b =o L X = NH, 0, 5, = N
& 6%
,Sl
VI.
EGG ALBUMIN
AND
323
THIOPHENE-%-AZLACTONE
The substituted protein is acidic in nature precipitating below pH 7 but remaining in solution at higher values of pH, indicating that the main mode of interaction is via the blocking of the amino groups of the protein. To determine whether linkages of other types between the thienylacrylic acid residues and the protein are formed, a study was carried out on samples of the protein with various degrees of substitution. The total thienylacrylic acid substitution in each sample was estimated by means of the ultraviolet absorption intensity in the regions of 275-280 rnp and 310-315 mp.3 The reaction with amino groups could TABLE Analytical
Sample no.
= I
Van Slyke data
-
N&m&in
_-
-mp./g. 0.0 0.0
1 2 3 4 5 6 7 8”
I Compound
Data on Egg Albumin-Thiophene&ada&me
No. of amino i:roups blockec 1
19 15 14 13.5 11 6.5 0
-
-
-
data IZ:7m,. 310-15 mp
275-80 mp
Electrophoretic mobilitiea x 10’
-24.8 19.6 13.5 9.3 8.0 5.3 3.2 0.2
23.1 20.6 17.0 13.7 13.3 11.6 9.6 7.1
19
1.6 1.8 2.1 3.0 4.7 7.2
-
ZXZ Ultraviolet _-I
cm./sec./a./cm. - 1.0
-0.99 -0.92
-0.81 -0.81 -0.79 -0.73 -0.71
-
a Pure egg albumin.
be followed by Van Slyke analysis of residual free amino groups. The electrophoretic analyses indicated the degree of change in the electrokinetic properties of the protein. The ultraviolet data are recorded in ~01s.4 and 5 of Table I. The Van a Sulfur analyses were carried out on all the samples. Although a definite gradation in sulfur content was found with increasing substitution, no quantitative conclusion could be reached, the values being low in all cases. When samples of egg albumin were subjected to the exact conditions of the azlactone introduction into it, it was found that their sulfur content had decreased. Thus, while native recrystallized egg albumin gave a sulfur content of 1.54yo, the treated control samples contained only 0.85-1.22~o. Such loss of sulfur atoms during this treatment at pH 8.0 and room temperature explains adequately the lack of agreement in the quantitative gradation of the sulfur values.
324
8. N. TIMASHEFF
AND
F. F. NORD
Slyke free amino group determinations and the electrophoretic mobilities are presented in ~01s.2, 3, and 6. The electrophoretic patterns -are shown in Fig. 2. From these data it can be seen that the total number of thienylacrylic acid residues introduced into the protein is not accounted for by the number of blocked amino groups for each particular sample, as the E:&. values increase after all the amino groups had reacted (samples
s&A 5,400 3,600 sec. sec. +DescendingFro. 2. Electrophoretic
A” 3,600 5,400 sec. sec. --Ascending-,
patterns of egg albumin-thiophene2azlactone PH = 7.5 (0.1 M phosphate buffer).
compound.
1 and 2). Therefore, it must be concluded that other types of interaction also occur, possibly with the serine or tyrosine hydroxyl, cysteine sulfhydryl, or proline secondary amino groups, all of which are known to be able to react with.azlactones. Furthermore, from data on samples 3, 4, and 5 it can be seen that while the total number of acrylic acid residues entering into the protein molecule is constantly increasing, the number of amino groups readily reacting seems to be limited to 70-75% of the total amino content. Complete saturation, however, of all amino groups takes place only in
VI.
EQG ALBUMIN
AND
THIOPHENE-2-AZLACTONE
325
cases of a large excess of adactone in the reaction mixture (samples 1 and 2). This could point to the presence of two types of amino groups in egg albumin, radically differing in the degree of reactivity, a fact which could also be deduced from results previously reported in the literature (14). The observed heterogeneity in the reactivity of amino groups of egg albumin could also be attributed to a possible difference in reactivity of the various fractions (15) found to be present in that protein. SUMMARY
A new modified protein was prepared by the reaction between egg albumin and a thiophene-2-azlactone. This acid polymer was shown by an ultraviolet absorption study to be a compound formed of egg albumin and a thienylacrylic acid. In this reaction, the principal reactive groups are the primary amino groups of the protein. However, other groups are also entering into competition, as only 70-75% of the amino groups were found to be strongly reactive. REFERENCES 1. BIER, M., AND Nom, F. F., Proc. Natl. Acad. Sci. U. S. 36, 17 (1949). 2. HERRIOTT, R. M., Advances in Protein Chem. 3, 170 (1947). H., AND 3. LEW&, H,, AND HAAG, R., 2. physiol. Chem. 266, 31 (1940); LEE, FERNHOLZ, M. E., ibid. 206, 37 (1940); LEYIYU%, H., BUCHHOLZ, K., AND FERNEOLZ, M. E., ibid. 267, 108 (1940). 4. CROWE, B. F., AND NORD, F. F., J. Org. Chem. 16, 81 (1950). 5. KING, W. J., AND Nom, F. F., J. Org. Chem. 13, 635 (1948). 6. FERGIER,M. F., AND VIQNEAUD, V. DU, J. Biol. Chem. 179, 61 (1949). 7. CROWE,B. F., AND Nom, F. F., J. Org. Chem. 15, 688 (1950). 8. TIMASAEFF, S. N., AND NORD, F. F., Arch. Biochem. Biophys. 31, 309 (1951). 9. CROWE,B. F., AND Nom, F. F., J. Org. Chem. 16, 1177 (1950). 10. GE&ACHER, C., AND GKJLBAS,G., Helv. Chim. Ada 10, 819 (1927). 11. VAN SLYKE, D. D., J. Biol. Chem. 16, 121 (1913). 12. MOORE, D. H., AND WHITE, J. U., Rev. Sci. ~netrumem% 16, 700 (1948). 13. LONQSWORTH, L. G., AND MACINNES, D. A., J. Am. Chem. Sot. 62,705 (1940). 14. KLECZKOWSIZI, A., &it. J. Ecptl. Path. 21, 1 (1940). 15. CANN, J. R., AND KIRKWOOD, J. G., Cold Spring Harbor Symposia 012Quant. Biol. 14, 9 (1950).