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A method for modification of carboxyl groups in proteins: Its application to the association of bovine β-lactoglobulin A
93
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 3512O A METHOD FOR MODIFICATION OF CARBOXYL GROUPS IN PROTEINS: ITS APPLICATION TO T H E ASSOCIATION OF BOVINE fi-LACTOGLOBULIN A J. McD. ARMSTRONG* AND H. A. M c K E N Z I E
Department of Physical Biochemistry, Institute of Advanced Studies, A ustralian National University, Canberra (Australia) (Received March 29th, 1967)
SUMMARY
A method for the modification of carboxyl groups in proteins is described. The water-soluble carbodiimide, I-cyclohexyl-3(2-morpholinyl-(4)-ethyl) carbodiimide metho-/%toluene sulphonate (CMC), is employed. The procedure is tested on bovine serum albumin as a model. It is applied to demonstrate the probable role of carboxyl groups in the dimeroctamer association of bovine fl-lactoglobulin A.
INTRODUCTION
Bovine fl-lactoglobulin A, which exists mainly as a dimer between pH 5.2 and pH 6, can polymerize near pH 4-7, at low temperature, to form predominantly an octamer of molecular weight i44ooo (refs. 1-3). The pH dependence of the association Js 'bell shaped' with a maximum near pH 4.5 and falling to 'zero' at pH's 3.5 and 5.2 (ref. 4)- The shape of this curve is typical of those for plots of the activity of an enzyme v s . pH value. The position of the maximum and the shape of the curve suggest the involvement of carboxyl groups (see also ref. 5). We report in the present paper a method for the modification of carboxyl groups in proteins, and the effect of this modification on the formation of octamers by bovine fl-lactoglobulin A. F R A E N K E L - C O N R A T AND OLCOTT 6 made an extensive study on the esterification of protein carboxyl groups with methanol-HC1. However, this method is not suitable in cases where the protein is easily denatured, and degradation may occur by way of an N-O acyl shift involving seryl and threonyl residues. It is known that the fi-lactoglobulins undergo eonformational changes in alcohol containing solvents. Esterification with diazomethane was generally less satisfactory than with methanol-HC1, and was not enirely specific. DOSCHER AND W I L C O X 7 used diazoacetamide to esterify ~chymotrypsinogen, however, competition for the reagent between the protein and water limits the extent of esterification which can be achieved. Carbodiimides have been used by SHEEHAN AND HLAVKAs in the preparation of Abbreviations : CMC, I-cyclohexyl-3 (2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluene sulphonate; BDC, N-benzyl-N'-3-dimethylaminopropylcarbodiimide. * Present address : Department of Biochemistry, Monash University, Clayton, Vic., Australia.
Biochim. Biophys. Acta, 147 (1967) 93-99
94
J. MCD. ARMSTRONG, H. A. MCKENZIE
peptides and for cross-linking gelatin by amide bridges. Their postulated reaction mechanism involves the formation of an isourea from the carbodiimide and a carboxyl group; this then reacts with an amine function to give the carboxamide and the urea corresponding to the carbodiimide. Water-soluble carbodiimides are commercially available, and the reaction can be carried out under mild conditions. We have used bovine serum albumin as a model for our studies before applying the method to fllactoglobulin A, since we wished to ascertain whether denaturation and cross-linking were likely to occur. An interesting short communication b y HOARE AND KOSHLAND9 has appeared since the present work was carried out. They describe a successful procedure for selective modification of carboxyl groups in proteins using N-benzyl-N'3-dimethylaminopropylcarbodiimide (BDC). MATERIALS AND METHODS
i-Cyclohexyl-3(2-morpholinyl-(4)-ethyl ) carbodiimide metho-p-toluene sulphonate (CMC) was obtained from Aldrich Chemical Co., Milwaukee, Wisc., U.S.A. Bovine serum albumin was from Sigma Chemicals, St. Louis, Mo., U.S.A. fl-Lactoglobulin A was prepared b y the method of ARMSTRONG,MCKENZIE AND SAWYER 1°.
General methods used were similar to those described previously 2. Absorption spectra were measured with a Cary Model 14 spectrophotometer (Applied Physics Corp., Monrovia, Calif.) using A ~ m , ~ 6.5I for bovine serum albumin, and 9.6 for fl-lactoglobulin A. The concentrations of the native and modified forms of bovine serum albumin were compared on the basis of equal absorbance in o.I M N a O H at 290 m/z, using a specific absorbance of 8. 7. Agreement between these results and the area under the schlieren patterns obtained from the ultracentrifuge was satisfactory. Modification of the proteins was carried out as follows: Both bovine serum albumin and fl-lactoglobulin have approx. 1.5 moles of ionizable carboxyl groups per IOOO g of protein. In a typical experiment calculated to give a m a x i m u m possible modification of 50 % of the total ionizable carboxyl groups, IOO/~moles of CMC was added to an aqueous solution containing I3o mg bovine serum albumin and Ioo /~moles of diethylammonium chloride, to give a final volume of 5 ml. The mixture, which was at p H 5-6, was allowed to stand for 9 ° nlin at room temperature and was then dialysed at 2-4 ° against 2 1 of distilled water for 4 ° h, with three changes of water. The characteristic benzenoid absorption spectrum of the p-toluene sulphonate ion was no longer observable, and it was assumed that dialysis was complete. Columns of Sephadex G-25 have been used to remove reagent in some experiments instead of dialysis, with buffers of o.I ionic strength. Paper electrophoresis was carried out in an L K B filter paper electrophoresis apparatus type 3276B, Schleicher and Schuell 2o43 B paper strips (LKB Produkter AB, Stockholm, Sweden), in either T r i s - E D T A buffer (pH 8.9) (60.5 g Tris, 6.0 g EDTA, 4.6 g H3BO 3 per 1; 0.5 M Tris, o.o2I M in EDTA, 0.075 M in H~BOa) or veronal buffer (pH 8.6) (20.6 g sodium diethylbarbiturate, 3.68 g diethylbarbituric acid per 1; I o.I). The strips were stained with bromthymol blue.
Biochim. Biophys..4cta, 147 (1967) 93-99
95
MODIFICATION OF CARBOXYL GROUPS
RESULTS
Serum albumin Samples of bovine serum albumin were reacted with different amounts of CMC. The modified samples migrated as discrete zones on paper electrophoresis at p H 8.6 or 8.9, and the rate of migration towards the anode decreased with increasing concentration of CMC used in the treatment. Thus the net negative charge on the protein decreased with increasing m a x i m u m possible percentage amidation of the total carboxyl groups present. If a considerable excess of CMC were used then a broad smear of protein about the origin was obtained, which is probably denatured protein. NH4C1 or (NH~)2S0 4 could be substituted for diethylammonium chloride. The pH of the reaction could be varied between 4 and 9 without effect on the observed migration of the modified protein. It was shown by sedimentation velocity experiments that the sedimentation coefficient of a number of modified bovine serum albumin samples in acetate buffer at p H 4.6 was not significantly changed b y modification. The boundary shapes for the modified proteins indicated no appreciable polymerization was occurring as a result of the modification. The absorption spectrum showed only minor changes (3-4 %) in intensity in the aromatic region, however, absorbance increased with increasing extent of modification in the region of 25o m~, as shown in Table I. The rotatory dispersion parameters a 0 and b0 for bovine serum albumin were not changed significantly b y the modification (Table I). A number of unsuccessful attempts were made to measure the amide content of modified bovine serum albumin where diethylamine was used in the reaction mixture. Increased amide N content was observed when ammonium salts were used. I t was noted that the pH value of solutions of modified bovine serum albumin exhaustively dialysed against water was higher than that of dialysed native protein, the p H increasing with the extent of modification. Similarly, the p H at which the protein precipitated on adding HC1 became higher with increasing extent of modification.
fl-Lactoglobulin A fi-Lactoglobulin A was treated with CMC under similar conditions to those for bovine serum albumin to give a m a x i m u m possible percentage modification of the carboxyl groups of 30 and 80. Sedimentation velocity measurements of the modifiod TABLE
I
EFFECT OF CARBOXYL GROUP MODIFICATION ON THE OPTICAL PROPERTIES OF BOVINE SERUM ALBUMIN
Maximum possible extent of modification (%)
A278mv/A255 m~ ao bo
0
20
60
2.1o --314 --304
1.89 --314 --296
1.6o --304 --3Ol
I00 1.4o --298 --290
Biochim. Biophys. Acta, 147 (1967) 9 3 - 9 9
96
J.
MCD. ARMSTRONG,
H. A. M C K E N Z I E
and native protein were made for sodium acetate-acetic acid solutions (I o.I) at pH 4.7 and 2.5 °. Typical patterns are shown in Fig. I. The native protein shows the bimodal pattern typical of the dimer-octamer reaction which it undergoes. The fast-moving peak has an s~0,w value of 5.5 S. The protein with a maximum possible of 3o % carboxyl modification shows a single peak, the maximum ordinate moving with an s20,w of approx. 2.6 S*. The pattern is symmetrical in general appearance in the early stages of sedimentation, but as time progresses some bimodal character becomes apparent. Although there is an appreciable amount of fast-moving material present it is considerably less than that of the unmodified protein. The pattern for the protein, in which a maximum of 8o % of the carboxyl groups have been modified, is also symmetrical in the early stages of sedimentation. However, in the later stages the boundary becomes somewhat skewed.
/l a
b
m
c
e
f
Fig. i. S e d i m e n t a t i o n p a t t e r n s of b o v i n e f l - l a c t o g l o b u l i n A a t p H 4.65 (r o . i , a c e t i c a c i d - s o d i u m a c e t a t e ) a t 2. 5 -Jz 2 ° w i t h a n d w i t h o u t m o d i f i c a t i o n of c a r b o x y l g r o u p s . S p e e d : 5 9 7 8 0 r e v . / m i n . a, b. N o n l o d i f i c a t i o n , c o n c n . 15 g/1. a, 64 r a i n a f t e r r e a c h i n g speed, b, i 2 o m i n . c, d. M a x i m u m 3 ° o, /o m o d i f i c a t i o n , c o n c n . 15 g/1. e, 68 rain. d, 134 m i n . e, f. M a x i m u m 8 0 % m o d i f i c a t i o n , e, ~4 m i n . f, 121 m i n . * T h e d i m e r f o r m of f l - l a c t o g l o b u l i n A h a s a n s2o,w of a p p r o x . protein.
Biochim. Bicphys. 3cta, 147 (1967) 9 3 - 9 9
2. 7 S for a i %
s o l u t i o n of
MODIFICATION OF CARBOXYL GROUPS
97
The m a x i m u m ordinate has an approx, s2o,w value of 2. 4 S and the weight average sedimentation coefficient, s20,w is 3.07 S. I t is evident that a m a x i m u m of 80 % modification of the carboxyl groups has largely prevented the dimer-octamer reaction from occurring. The ultraviolet absorption spectrum of the modified protein was unchanged in the aromatic region, but changes occurred in the short wavelength side of the 280 my, similar to those observed for bovine serum albumin. It will be recalled from the DISCUSSION in the preceding paper 4 that the MOFFITT-YAxG plots of the optical rotatory dispersion data of the fl-lactoglobulins indicate some departure from linearity below 330 mt~ at p H 4.7. The deviation is temperature dependent and is moderate at 3 o°. The value of Ec~157s, a 0 and b0 for the native protein are approx. --29% --14 o°, --60 °. There is only a small change in these values for the protein with 3o % maxim u m carboxyl modification. In the case of the protein with 8o % m a x i m u m carboxyl modification there was a more pronounced change in optical rotatory properties, Ea157s, a 0 and b0 becoming approx. --36°, - - I 9 o°, --55 °. DISCUSSION
The decrease in electrophoretic migration of bovine serum albumin towards the anode after modification with CMC provides evidence that the net negative charge of the protein at p H 8.6-8. 9 has been decreased, in approximate proportion to the m a x i m u m possible extent of modification. It is also clear that complete blockage of the carboxyl groups has not been achieved. Further evidence for loss of ionizable carboxyl groups is the increase in the isoionic point of the protein after modification. The main evidence for retention of conformation after blocking the carboxyl groups is the virtually unchanged rotatory behaviour of bovine serum albumin after reaction with CMC. The small changes in the absorption spectrum of modified bovine serum albumin probably reflect the change in perturbation of aromatic residues due to charge (see, e.g. ref. II), rather than increased exposure of the aromatic residues following conformational changes. The increase in absorption of the modified bovine serum albumin in the region of 250 m~ is difficult to explain, since it occurs at rather too long wavelengths to arise from end absorption due to amide. The sedimentation experiments with bovine serum albumin indicate that the hydrodynamic behaviour of the protein at p H 4.6 has not been altered appreciably b y modification, which is consistent with the lack of conformational change, as indicated b y the optical rotatory dispersion measurements. The absence of heavy components shows that aggregation due to amide cross links did not take place. The nature of the modification to the carboxyl groups is not clear. We had assumed that the intermediate isourea derivative was converted to the amide, however, an O-N shift could occur, resulting in a urea substituent on the carboxyl groups. Such a derivative was obtained by IV[UKERJEE AND SRI RAM12, who reacted bovine serum albumin with I-ethyl-3(3-dimethylaminopropyl) carbodiimide. They observed that some aggregation of the protein had occurred, presumably from the formation of intermolecular amide bridges. P, IEHM AND SCHERAGA1:~ used CMC to modify the carboxyl groups of ribonuclease. In both cases, the earbodiimide and protein were the only reactants present. Although the products of the modification were not identified, it seems probable that the protein carboxyl groups were converted to the Biochim. Biophys. Acta, 147 (1967) 93-99
98
J. MCD. ARMSTRONG, H. A. MCKENZIE
acyl urea derivatives (II). RIEHM AND SCHERAGA suggest that the unstable isourea intermediate m a y rearrange also to an imide. O
Ji
Pr-C-N-R Isourea (I) ~
O=C
I ttN-R'
(II)
In the present studies and those of HOARE AND KOSHLAND9, a reagent with an amine function was included in the reaction mixture. HOARE AND KOSHLAND present evidence that the reaction with the carbodiimide under their conditions is N-R Pr-C00H
+ C
HN-R H+ --~
Pr-C00-C
I
N-R'
HN-R' _
(I)
/
HN-R
0
YNH,, {! ' ---> P r - C - N H Y + 0 = C - .
H+
HN-R' (III)
They demonstrated the incorporation of YNH~ into protein by this reaction. Since we observed increased amide N in bovine serum albumin after reaction with CMC and ammonium salts, it seems that the amide derivative (III) was produced under our conditions. While the evidence which we have obtained on the involvement of carboxyl groups in the formation of octamers of bovine fl-lactoglobulin A is not conclusive, it suggests strongly that they are involved. It is not possible to state on the basis of the optical rotatory dispersion measurements whether the changes in the 8o % (max.) modified protein are due to conformational or other changes in the molecule. However, the conformation of the 30 % (max.) modified sample had not changed but its ability to associate to the octamer was markedly decreased. Comparative studies on fl-lactoglobulins from various mammals in this laboratory (see ref. I) have shown that only bovine fi-lactoglobulin A undergoes this association reaction, and that it is the only one in which an additional aspartyl residue per chain (I8 ooo monomer) is found. Of interest in this connexion is the isolation and examination by BELL and coworkers14,15 of a new fl-lactoglobulin variant, fl-lactoglobulinDroughtmaster, since the present work. This variant does not form octamers. It has the same amino acid composition as the bovine A variant, but it has sialic acid and hexosamine groups attached to it. These groups occur in a peptide containing inter alia, arginine, serine and glutamic acids, and not in the peptide containing the extra aspartic acid residue. The carbohydrate moiety prevents the octamerization reaction occurring either by direct blockage of essential carboxyl groups (e.g. glutamic acid for aspartic-glutamic carboxyl hydrogen bonds), or by steric interference. ACKNOWLEDGEMENT
Appreciation is expressed to the Australian Dairy Board for assistance in this work and the opportunity to present an account of it at the Board's Research Seminar, June I964 . Biochim, B i o p h y s . . 4 c t a , 147 (1967) 93-99
MODIFICATION OF CARBOXYL GROUPS
9~
REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15
H. A. McKENzlE, Advan. Protein Chem., 22 (1967) 55. H. A. MCKENZlE, W. H. SAWYER AND M. B. SMITH, Biochim. Biophys. Acta, 147 (1967) 73. R. TOWNEND AND S. N, TIMASHEFF, J. Am. Chem. Soc., 82 (196o) 3168. R. TOWNEND AND S. N. TIMASHEFF, Arch. Biochem. Biophys., 63 (1956) 482. S. N. TIMASHEFF AND R. TOWNEND, J. Dairy Sci., 45 (1962) 259. H. FRAENKEL-CONRAT AND H. S. OLCOTT, J. Biol. Chem., 161 (1945) 259. M. S. D0SCHER AND P. E. WILCOX, J. Biol. Chem., 236 (1961) 1328. J. C. SHEEHAN AND J. J. HLAVKA, J . Am. Chem. Soc., 79 (1957) 4528. D. G. HOARE AND D. E. KOSHLAND, JR., J. Am. Chem. Soc., 88 (1966) 2057. J. McD. ARMSTRONG, H. A. MCKENZlE AND W. H. SAWYER, Biochim. Biophys. Acta, 147 (1967) 6o. D. ]3. WETLAUPER, J. T. EDSALL AND ]3. R. HOLLINGWORTH, J. Biol. Chem., 233 (1958) 1421. H. MUKERJEE AND J. SRI RAM, Federation Proc., 24 (1965) 418. J. P. RIEHM AND H. A. SCHERAGA, Biochemistry, 5 (1966) 99. K . ]3ELL, H. A. McK~I~ZlE AND W. H. MURPHY, Australian J. Sci., 29 (1966) 87. K. BELL, H. A. McKENzlE AND D. C. SHAW, Australian f . Sci., 29 (1966) 86.
Bicchim. Biophys. Acta, 147 (1967) 9 3 - 9 9
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 3512O A METHOD FOR MODIFICATION OF CARBOXYL GROUPS IN PROTEINS: ITS APPLICATION TO T H E ASSOCIATION OF BOVINE fi-LACTOGLOBULIN A J. McD. ARMSTRONG* AND H. A. M c K E N Z I E
Department of Physical Biochemistry, Institute of Advanced Studies, A ustralian National University, Canberra (Australia) (Received March 29th, 1967)
SUMMARY
A method for the modification of carboxyl groups in proteins is described. The water-soluble carbodiimide, I-cyclohexyl-3(2-morpholinyl-(4)-ethyl) carbodiimide metho-/%toluene sulphonate (CMC), is employed. The procedure is tested on bovine serum albumin as a model. It is applied to demonstrate the probable role of carboxyl groups in the dimeroctamer association of bovine fl-lactoglobulin A.
INTRODUCTION
Bovine fl-lactoglobulin A, which exists mainly as a dimer between pH 5.2 and pH 6, can polymerize near pH 4-7, at low temperature, to form predominantly an octamer of molecular weight i44ooo (refs. 1-3). The pH dependence of the association Js 'bell shaped' with a maximum near pH 4.5 and falling to 'zero' at pH's 3.5 and 5.2 (ref. 4)- The shape of this curve is typical of those for plots of the activity of an enzyme v s . pH value. The position of the maximum and the shape of the curve suggest the involvement of carboxyl groups (see also ref. 5). We report in the present paper a method for the modification of carboxyl groups in proteins, and the effect of this modification on the formation of octamers by bovine fl-lactoglobulin A. F R A E N K E L - C O N R A T AND OLCOTT 6 made an extensive study on the esterification of protein carboxyl groups with methanol-HC1. However, this method is not suitable in cases where the protein is easily denatured, and degradation may occur by way of an N-O acyl shift involving seryl and threonyl residues. It is known that the fi-lactoglobulins undergo eonformational changes in alcohol containing solvents. Esterification with diazomethane was generally less satisfactory than with methanol-HC1, and was not enirely specific. DOSCHER AND W I L C O X 7 used diazoacetamide to esterify ~chymotrypsinogen, however, competition for the reagent between the protein and water limits the extent of esterification which can be achieved. Carbodiimides have been used by SHEEHAN AND HLAVKAs in the preparation of Abbreviations : CMC, I-cyclohexyl-3 (2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluene sulphonate; BDC, N-benzyl-N'-3-dimethylaminopropylcarbodiimide. * Present address : Department of Biochemistry, Monash University, Clayton, Vic., Australia.
Biochim. Biophys. Acta, 147 (1967) 93-99
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J. MCD. ARMSTRONG, H. A. MCKENZIE
peptides and for cross-linking gelatin by amide bridges. Their postulated reaction mechanism involves the formation of an isourea from the carbodiimide and a carboxyl group; this then reacts with an amine function to give the carboxamide and the urea corresponding to the carbodiimide. Water-soluble carbodiimides are commercially available, and the reaction can be carried out under mild conditions. We have used bovine serum albumin as a model for our studies before applying the method to fllactoglobulin A, since we wished to ascertain whether denaturation and cross-linking were likely to occur. An interesting short communication b y HOARE AND KOSHLAND9 has appeared since the present work was carried out. They describe a successful procedure for selective modification of carboxyl groups in proteins using N-benzyl-N'3-dimethylaminopropylcarbodiimide (BDC). MATERIALS AND METHODS
i-Cyclohexyl-3(2-morpholinyl-(4)-ethyl ) carbodiimide metho-p-toluene sulphonate (CMC) was obtained from Aldrich Chemical Co., Milwaukee, Wisc., U.S.A. Bovine serum albumin was from Sigma Chemicals, St. Louis, Mo., U.S.A. fl-Lactoglobulin A was prepared b y the method of ARMSTRONG,MCKENZIE AND SAWYER 1°.
General methods used were similar to those described previously 2. Absorption spectra were measured with a Cary Model 14 spectrophotometer (Applied Physics Corp., Monrovia, Calif.) using A ~ m , ~ 6.5I for bovine serum albumin, and 9.6 for fl-lactoglobulin A. The concentrations of the native and modified forms of bovine serum albumin were compared on the basis of equal absorbance in o.I M N a O H at 290 m/z, using a specific absorbance of 8. 7. Agreement between these results and the area under the schlieren patterns obtained from the ultracentrifuge was satisfactory. Modification of the proteins was carried out as follows: Both bovine serum albumin and fl-lactoglobulin have approx. 1.5 moles of ionizable carboxyl groups per IOOO g of protein. In a typical experiment calculated to give a m a x i m u m possible modification of 50 % of the total ionizable carboxyl groups, IOO/~moles of CMC was added to an aqueous solution containing I3o mg bovine serum albumin and Ioo /~moles of diethylammonium chloride, to give a final volume of 5 ml. The mixture, which was at p H 5-6, was allowed to stand for 9 ° nlin at room temperature and was then dialysed at 2-4 ° against 2 1 of distilled water for 4 ° h, with three changes of water. The characteristic benzenoid absorption spectrum of the p-toluene sulphonate ion was no longer observable, and it was assumed that dialysis was complete. Columns of Sephadex G-25 have been used to remove reagent in some experiments instead of dialysis, with buffers of o.I ionic strength. Paper electrophoresis was carried out in an L K B filter paper electrophoresis apparatus type 3276B, Schleicher and Schuell 2o43 B paper strips (LKB Produkter AB, Stockholm, Sweden), in either T r i s - E D T A buffer (pH 8.9) (60.5 g Tris, 6.0 g EDTA, 4.6 g H3BO 3 per 1; 0.5 M Tris, o.o2I M in EDTA, 0.075 M in H~BOa) or veronal buffer (pH 8.6) (20.6 g sodium diethylbarbiturate, 3.68 g diethylbarbituric acid per 1; I o.I). The strips were stained with bromthymol blue.
Biochim. Biophys..4cta, 147 (1967) 93-99
95
MODIFICATION OF CARBOXYL GROUPS
RESULTS
Serum albumin Samples of bovine serum albumin were reacted with different amounts of CMC. The modified samples migrated as discrete zones on paper electrophoresis at p H 8.6 or 8.9, and the rate of migration towards the anode decreased with increasing concentration of CMC used in the treatment. Thus the net negative charge on the protein decreased with increasing m a x i m u m possible percentage amidation of the total carboxyl groups present. If a considerable excess of CMC were used then a broad smear of protein about the origin was obtained, which is probably denatured protein. NH4C1 or (NH~)2S0 4 could be substituted for diethylammonium chloride. The pH of the reaction could be varied between 4 and 9 without effect on the observed migration of the modified protein. It was shown by sedimentation velocity experiments that the sedimentation coefficient of a number of modified bovine serum albumin samples in acetate buffer at p H 4.6 was not significantly changed b y modification. The boundary shapes for the modified proteins indicated no appreciable polymerization was occurring as a result of the modification. The absorption spectrum showed only minor changes (3-4 %) in intensity in the aromatic region, however, absorbance increased with increasing extent of modification in the region of 25o m~, as shown in Table I. The rotatory dispersion parameters a 0 and b0 for bovine serum albumin were not changed significantly b y the modification (Table I). A number of unsuccessful attempts were made to measure the amide content of modified bovine serum albumin where diethylamine was used in the reaction mixture. Increased amide N content was observed when ammonium salts were used. I t was noted that the pH value of solutions of modified bovine serum albumin exhaustively dialysed against water was higher than that of dialysed native protein, the p H increasing with the extent of modification. Similarly, the p H at which the protein precipitated on adding HC1 became higher with increasing extent of modification.
fl-Lactoglobulin A fi-Lactoglobulin A was treated with CMC under similar conditions to those for bovine serum albumin to give a m a x i m u m possible percentage modification of the carboxyl groups of 30 and 80. Sedimentation velocity measurements of the modifiod TABLE
I
EFFECT OF CARBOXYL GROUP MODIFICATION ON THE OPTICAL PROPERTIES OF BOVINE SERUM ALBUMIN
Maximum possible extent of modification (%)
A278mv/A255 m~ ao bo
0
20
60
2.1o --314 --304
1.89 --314 --296
1.6o --304 --3Ol
I00 1.4o --298 --290
Biochim. Biophys. Acta, 147 (1967) 9 3 - 9 9
96
J.
MCD. ARMSTRONG,
H. A. M C K E N Z I E
and native protein were made for sodium acetate-acetic acid solutions (I o.I) at pH 4.7 and 2.5 °. Typical patterns are shown in Fig. I. The native protein shows the bimodal pattern typical of the dimer-octamer reaction which it undergoes. The fast-moving peak has an s~0,w value of 5.5 S. The protein with a maximum possible of 3o % carboxyl modification shows a single peak, the maximum ordinate moving with an s20,w of approx. 2.6 S*. The pattern is symmetrical in general appearance in the early stages of sedimentation, but as time progresses some bimodal character becomes apparent. Although there is an appreciable amount of fast-moving material present it is considerably less than that of the unmodified protein. The pattern for the protein, in which a maximum of 8o % of the carboxyl groups have been modified, is also symmetrical in the early stages of sedimentation. However, in the later stages the boundary becomes somewhat skewed.
/l a
b
m
c
e
f
Fig. i. S e d i m e n t a t i o n p a t t e r n s of b o v i n e f l - l a c t o g l o b u l i n A a t p H 4.65 (r o . i , a c e t i c a c i d - s o d i u m a c e t a t e ) a t 2. 5 -Jz 2 ° w i t h a n d w i t h o u t m o d i f i c a t i o n of c a r b o x y l g r o u p s . S p e e d : 5 9 7 8 0 r e v . / m i n . a, b. N o n l o d i f i c a t i o n , c o n c n . 15 g/1. a, 64 r a i n a f t e r r e a c h i n g speed, b, i 2 o m i n . c, d. M a x i m u m 3 ° o, /o m o d i f i c a t i o n , c o n c n . 15 g/1. e, 68 rain. d, 134 m i n . e, f. M a x i m u m 8 0 % m o d i f i c a t i o n , e, ~4 m i n . f, 121 m i n . * T h e d i m e r f o r m of f l - l a c t o g l o b u l i n A h a s a n s2o,w of a p p r o x . protein.
Biochim. Bicphys. 3cta, 147 (1967) 9 3 - 9 9
2. 7 S for a i %
s o l u t i o n of
MODIFICATION OF CARBOXYL GROUPS
97
The m a x i m u m ordinate has an approx, s2o,w value of 2. 4 S and the weight average sedimentation coefficient, s20,w is 3.07 S. I t is evident that a m a x i m u m of 80 % modification of the carboxyl groups has largely prevented the dimer-octamer reaction from occurring. The ultraviolet absorption spectrum of the modified protein was unchanged in the aromatic region, but changes occurred in the short wavelength side of the 280 my, similar to those observed for bovine serum albumin. It will be recalled from the DISCUSSION in the preceding paper 4 that the MOFFITT-YAxG plots of the optical rotatory dispersion data of the fl-lactoglobulins indicate some departure from linearity below 330 mt~ at p H 4.7. The deviation is temperature dependent and is moderate at 3 o°. The value of Ec~157s, a 0 and b0 for the native protein are approx. --29% --14 o°, --60 °. There is only a small change in these values for the protein with 3o % maxim u m carboxyl modification. In the case of the protein with 8o % m a x i m u m carboxyl modification there was a more pronounced change in optical rotatory properties, Ea157s, a 0 and b0 becoming approx. --36°, - - I 9 o°, --55 °. DISCUSSION
The decrease in electrophoretic migration of bovine serum albumin towards the anode after modification with CMC provides evidence that the net negative charge of the protein at p H 8.6-8. 9 has been decreased, in approximate proportion to the m a x i m u m possible extent of modification. It is also clear that complete blockage of the carboxyl groups has not been achieved. Further evidence for loss of ionizable carboxyl groups is the increase in the isoionic point of the protein after modification. The main evidence for retention of conformation after blocking the carboxyl groups is the virtually unchanged rotatory behaviour of bovine serum albumin after reaction with CMC. The small changes in the absorption spectrum of modified bovine serum albumin probably reflect the change in perturbation of aromatic residues due to charge (see, e.g. ref. II), rather than increased exposure of the aromatic residues following conformational changes. The increase in absorption of the modified bovine serum albumin in the region of 250 m~ is difficult to explain, since it occurs at rather too long wavelengths to arise from end absorption due to amide. The sedimentation experiments with bovine serum albumin indicate that the hydrodynamic behaviour of the protein at p H 4.6 has not been altered appreciably b y modification, which is consistent with the lack of conformational change, as indicated b y the optical rotatory dispersion measurements. The absence of heavy components shows that aggregation due to amide cross links did not take place. The nature of the modification to the carboxyl groups is not clear. We had assumed that the intermediate isourea derivative was converted to the amide, however, an O-N shift could occur, resulting in a urea substituent on the carboxyl groups. Such a derivative was obtained by IV[UKERJEE AND SRI RAM12, who reacted bovine serum albumin with I-ethyl-3(3-dimethylaminopropyl) carbodiimide. They observed that some aggregation of the protein had occurred, presumably from the formation of intermolecular amide bridges. P, IEHM AND SCHERAGA1:~ used CMC to modify the carboxyl groups of ribonuclease. In both cases, the earbodiimide and protein were the only reactants present. Although the products of the modification were not identified, it seems probable that the protein carboxyl groups were converted to the Biochim. Biophys. Acta, 147 (1967) 93-99
98
J. MCD. ARMSTRONG, H. A. MCKENZIE
acyl urea derivatives (II). RIEHM AND SCHERAGA suggest that the unstable isourea intermediate m a y rearrange also to an imide. O
Ji
Pr-C-N-R Isourea (I) ~
O=C
I ttN-R'
(II)
In the present studies and those of HOARE AND KOSHLAND9, a reagent with an amine function was included in the reaction mixture. HOARE AND KOSHLAND present evidence that the reaction with the carbodiimide under their conditions is N-R Pr-C00H
+ C
HN-R H+ --~
Pr-C00-C
I
N-R'
HN-R' _
(I)
/
HN-R
0
YNH,, {! ' ---> P r - C - N H Y + 0 = C - .
H+
HN-R' (III)
They demonstrated the incorporation of YNH~ into protein by this reaction. Since we observed increased amide N in bovine serum albumin after reaction with CMC and ammonium salts, it seems that the amide derivative (III) was produced under our conditions. While the evidence which we have obtained on the involvement of carboxyl groups in the formation of octamers of bovine fl-lactoglobulin A is not conclusive, it suggests strongly that they are involved. It is not possible to state on the basis of the optical rotatory dispersion measurements whether the changes in the 8o % (max.) modified protein are due to conformational or other changes in the molecule. However, the conformation of the 30 % (max.) modified sample had not changed but its ability to associate to the octamer was markedly decreased. Comparative studies on fl-lactoglobulins from various mammals in this laboratory (see ref. I) have shown that only bovine fi-lactoglobulin A undergoes this association reaction, and that it is the only one in which an additional aspartyl residue per chain (I8 ooo monomer) is found. Of interest in this connexion is the isolation and examination by BELL and coworkers14,15 of a new fl-lactoglobulin variant, fl-lactoglobulinDroughtmaster, since the present work. This variant does not form octamers. It has the same amino acid composition as the bovine A variant, but it has sialic acid and hexosamine groups attached to it. These groups occur in a peptide containing inter alia, arginine, serine and glutamic acids, and not in the peptide containing the extra aspartic acid residue. The carbohydrate moiety prevents the octamerization reaction occurring either by direct blockage of essential carboxyl groups (e.g. glutamic acid for aspartic-glutamic carboxyl hydrogen bonds), or by steric interference. ACKNOWLEDGEMENT
Appreciation is expressed to the Australian Dairy Board for assistance in this work and the opportunity to present an account of it at the Board's Research Seminar, June I964 . Biochim, B i o p h y s . . 4 c t a , 147 (1967) 93-99
MODIFICATION OF CARBOXYL GROUPS
9~
REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15
H. A. McKENzlE, Advan. Protein Chem., 22 (1967) 55. H. A. MCKENZlE, W. H. SAWYER AND M. B. SMITH, Biochim. Biophys. Acta, 147 (1967) 73. R. TOWNEND AND S. N, TIMASHEFF, J. Am. Chem. Soc., 82 (196o) 3168. R. TOWNEND AND S. N. TIMASHEFF, Arch. Biochem. Biophys., 63 (1956) 482. S. N. TIMASHEFF AND R. TOWNEND, J. Dairy Sci., 45 (1962) 259. H. FRAENKEL-CONRAT AND H. S. OLCOTT, J. Biol. Chem., 161 (1945) 259. M. S. D0SCHER AND P. E. WILCOX, J. Biol. Chem., 236 (1961) 1328. J. C. SHEEHAN AND J. J. HLAVKA, J . Am. Chem. Soc., 79 (1957) 4528. D. G. HOARE AND D. E. KOSHLAND, JR., J. Am. Chem. Soc., 88 (1966) 2057. J. McD. ARMSTRONG, H. A. MCKENZlE AND W. H. SAWYER, Biochim. Biophys. Acta, 147 (1967) 6o. D. ]3. WETLAUPER, J. T. EDSALL AND ]3. R. HOLLINGWORTH, J. Biol. Chem., 233 (1958) 1421. H. MUKERJEE AND J. SRI RAM, Federation Proc., 24 (1965) 418. J. P. RIEHM AND H. A. SCHERAGA, Biochemistry, 5 (1966) 99. K . ]3ELL, H. A. McK~I~ZlE AND W. H. MURPHY, Australian J. Sci., 29 (1966) 87. K. BELL, H. A. McKENzlE AND D. C. SHAW, Australian f . Sci., 29 (1966) 86.
Bicchim. Biophys. Acta, 147 (1967) 9 3 - 9 9