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Introduction of sulfhydryl groups into proteins at carboxyl sites
382
Biochimica et Biophysica Acta, 1038 (1990) 382-385
Elsevier BBAPRO 33636
Introduction of sulfhydryl groups into proteins at carboxyl sites * C h a o m e i Lin, K e v i n A. Mihal * * a n d Rick J. K r u e g e r Department of Biochemistry and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE (U.S.A)
(Received 18 December1989)
Key words: Sulfbydrylgroup; Protein modification;Carbodiimide A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.
Introduction Incorporation of sulfhydryl groups into proteins is frequently employed to facilitate formation of covalent crosslinks to other proteins or labeling reagents. The variety of reagents available which react rapidly and specifically with sulfhydryl groups makes them the group of choice in many cases. While sulfhydryl group incorporation at various protein functional groups has been studied [1], derivatization of amino groups has been employed most frequently (e.g. Refs. 2 and 3). The development of water soluble carbodiimides [4] has permitted relatively selective incorporation of amines at protein carboxyl groups. Fattom et al. [5] have described a procedure for incorporating sulfhydryl groups at carboxyl sites of carbohydrates. This two-step procedure utilized ECD mediated incorporation of cystamine into N-acetylmannoseaminouronic acid to form an amide bond between the amino group of cystamine and the carboxyl group of the carbohydrate. The disulfide was then reduced to yield a carbohydrate containing a cysteamide sulfhydryl group. * Agricultural Research Division, University of Nebraska Journal Series No. 9102. * * Present address: Department of Biochemistry,Boston University School of Medicine, Boston, MA 02118, U.S.A. Abbreviations: ECD, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ACTH, adrenocorticotropichormone; EDs0, dose resulting in 50% of maximal response; SDS, sodium dodecyl sulfate. Correspondence: R.J. Krueger, Room 314, Department of Biochemistry, Universityof Nebraska-Lincoln,Lincoln,NE 68583-0718,U.S.A.
We show here that this sulfhydryl group incorporation scheme can be used to introduce sulfhydryl groups directly into proteins. The procedure was utilized to make thiolated enzymes and polypeptide hormones that retained full biological activity. Materials and Methods Materials
ECD, cystamine, dithiothreitol, bovine carbonic anhydrase, p-nitrophenyl acetate, and porcine ACTH were purchased from Sigma Chemical Co. Carbonic anhydrase was examined by SDS-polyacrylamide gel electrophoresis [6] and appeared to be better than 99% homogeneous. A C T H was purified by ion-exchange chromatography on Whatman CM-52 cellulose [7] and by preparative reversed phase HPLC on a Baker 15 # m 1 x 25 cm C18 wide pore colunm. E C D mediated incorporation of cystamine into protein
Incorporation of cystamine into proteins was carried out at 2 2 ° C in 0.5 M cystamine, 0.1 M Mes-NaOH (pH 4.2) unless otherwise noted. Carbonic anhydrase and A C T H concentrations were 0.2 mM. The reaction was initiated by the addition of ECD. The carbonic anhydrase modification reaction was 30 min in length, and the derivatized protein was separated from excess cystamine by precipitation with ammonium sulfate (added to 80% of saturation). The precipitate was collected by centrifugation and dissolved in 50 mM Tris-HC1 (pH 7.0). Dithiothreitol was added to a final concentration of 6 mM, and the sample was incubated for 20 min at room temperature to reduce the incorpo-
0167-4838/90/$03.50 © 1990 Elsevier SciencePubfishers B.V. (BiomedicalDivision)
383 rated disulfide groups to the sulfhydryl form. After reduction, the protein was precipitated with a m m o n i u m sulfate twice as described above to separate the carbonic anhydrase derivative from dithiothreitol and 2aminoethanethiol that was released from the protein by reduction. T h e derived protein was dissolved in 0.1 M Tris-HC1 and assayed as described below. The reaction conditions for sulfhydryl group incorporation into A C T H were similar to those for carbonic anhydrase except that the ECD concentration was 2 m M unless otherwise noted. Products from the incorporation of cystamine into A C T H were separated by chromatography on a Sephadex G-10 column equilibrated with 0.1 M acetic acid, and peptides obtained from this separation were reduced with dithiothreitol as above. The products were then subjected to reversed phase H P L C on a Bakerbond C18 wide pore 5 #m, 4.6 × 250 m m column using linear gradients of 0.05% trifluoroacetic acid (solvent A) and 0.05% trifluoroacetic acid, 50% acetonitrile (solvent B). The peptide fractions collected from H P L C separations were taken to dryness under vacuum, and analyzed as described below.
Analysis of modified proteins Protein concentrations of carbonic anhydrase [8] and A C T H [9] were monitored by ultraviolet absorption spectroscopy. Sulfhydryl group content of proteins was measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) [10]. Carbonic anhydrase activity was measured by a p-nitrophenyl acetate esterase assay [11], which was conducted in 0.1 M Tris-HC1 ( p H 7.4). The biological activity of A C T H was assessed by the capacity of peptides to stimulate steroidogenesis in suspensions of rat adrenocortical cells prepared as described by Ray and Strott [12]. Cell suspensions were incubated with a range of concentrations of peptide for 1 h and assayed for fluorogenic steroid content [13]. Peptide dose vs. steroidogenic response curves were plotted, and the peptide concentration yielding 50% of the maximal steroidogenic rate (EDs0) was determined. Results
The influence of carbodiimide concentration on sulfhydryl group incorporation was examined for carbonic anhydrase in the 1 to 100 m M E C D range and for A C T H in the 2.5 to 15 m M E C D range at p H 4.2. Increasing the E C D concentration caused an increase in the number of cystamine molecules incorporated per protein molecule. The sulfhydryl group content was determined after reduction of the covalently bound, cystamine derived disulfide to the sulfhydryl form (Fig. 1). The degree of sulfhydryl group incorporation is expressed as the percentage of the m a x i m u m possible, assuming the incorporation of one sulfhydryl group per carboxyl site, 32 carboxyl groups per carbonic anhydrase
E 70 E
/
60
50
/
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40
0
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o
& ,30 L 0
20 i 10, I 0 0.0
I
I
I
I
0.5
1.0
1.5
2.0
log [ECDI ( m M ) ] Fig. 1. ECD concentration dependence of incorporation of sulfhydryl groups into carbonic anhydrase and ACTH. Modified carbonic anhydrase and ACTH were prepared as described in Materials and Methods. Percent maximal incorporation is based on 32 carboxyi groups per carbonic anhydrase molecule and 7 carboxyl groups per ACTH molecule, o, carbonic anhydrase; O, ACTH. molecule [8] and 7 carboxyl groups per A C T H [14]. The highest levels of sulfhydryl group incorporation observed were 22 sulfhydryl groups per carbonic anhydrase molecule at 100 m M ECD and 2.1 sulfhydryl groups per A C T H molecule at 15 m M ECD. Ultraviolet absorption spectra [9] of A C T H derivatives at this level of sulfhydryl group incorporation showed no reduction in the tyrosine to tryptophan ratio, indicating that tyrosine residues had not reacted with E C D to form O-aryl isourea adducts [15]. Because m a n y proteins are not stable at p H 4.2, the incorporation of sulfhydryl groups was determined at higher p H values with carbonic anhydrase. A striking decrease in sulfhydryl group incorporation was observed (Table I). This result was anticipated because the carboxylic acid rather than the carboxylate ion is usually considered to be the reactive species [16]. Analysis of the p-nitrophenyl acetate esterase activity of modified carbonic anhydrase showed that introduction of up TABLE I Effect of pH on sulfhydryl group incorporation into carbonic anhydrase. ECD (100 mM) mediated reaction of cystamine with carbonic anhydrase, reduction of the incorporated cystamine disulfides to free thiols, and determination of protein concentration, sulfhydryl group content, and esterase activity were carried out as described in Materials and Methods. Reactions at pH 4.2 and 5.0 were carried out in 0.1 M Mes-NaOH. The reaction at pH 6.0 was carried out in 0.1 M potassium phosphate, and the reaction at pH 7.4, which lacked ECD, was in 0.1 M Tris-HC1. pH 4.2 5.0 6.0 7.4 (no ECD)
mol -SH/mol carbonic anhydrase 22.1 8.8 2.1 0
Esterase activity (%) 87 100 98 100
384
1
E C
0 v W
o
3 4
6
a,, o t/]
I
I
I
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I
I
I
10
15
20
25
30
35
TIME (min) Fig. 2. Separation of sulfhydryl containing ACTH peptides by C18 HPLC. Incorporation of cystamine into ACTH at 2 mM ECD and separation of modified peptide from excess cystamine was carried out as described in Materials and Methods. The modified peptide was then reduced with 0.1 M dithiothreitol, and the sulfhydryl containing ACTH peptides were separated by C18 HPLC. The bars on the chromatogram indicate where fraction collection was initiated and terminated.
to nine sulflaydryl groups per molecule had very little effect on enzymatic activity, and even at 22 sulfhydryl groups per molecule, the inhibitory effect was slight
(13%). Porcine ACTH contains seven carboxyl groups. Of these only one, Glu 5, is thought to be directly involved in adrenocortical cell ACTH receptor binding associated with stimulation of steroidogenesis [17]. To obtain ACTH peptides with minimal loss of receptor binding activity, a sulfhydryl group incorporation reaction was carried out at a low ECD concentration to generate a peptide population that contained an average of 0.3 sulfhydryl groups per molecule. The object was to limit modification to at most one carboxyl group per ACTH molecule. Peptide products were then reduced to their sulfhydryl form and resolved by C18 HPLC (Fig. 2). TABLE II Sulfbydryl group content and steroidogenic potency of modified ACTH species. Preparation of sulfhydryl containing ACTH peptides, purification, and their analysis are described in Materials and Methods. The fraction numbers refer to the peaks indicated in Fig. 2. The EDs0 associated with peak 1 was equal to that of native ACTH. Fraction No.
mol -SH/mol ACTH
EDs0 (nM)
1 2 3 4 5 6 7 8
0.02 0.69 0.98 1.06 1.01 1.15 0.94 0.32
0.16 0.25 0.13 0.19 0.13 0.16 1.26 3.97
The peptides eluting at and after the unmodified form of ACTH (peak 1) were collected and analyzed for peptide and sulfhydryl content as well as capacity to stimulate steroidogenesis in suspensions of rat adrenocortical cells. Seven modified peptide fractions were obtained. The yield of sulflaydryl containing peptide (peaks 2 through 8) was 25% of the total peptide eluted from the C18 column. Five of these fractions (peaks 3-7) contained approximately one sulfhydryl group per peptide molecule, while non-integer values were obtained for peaks 2 and 8 (Table II). Peptide peaks 2 through 8 all stimulated steroidogenesis to the same maximal level observed with native ACTH. However, while dose-response analyses showed that peaks 2 to 6 had EDs0 values close to that of native ACTH, two of the peaks (7 and 8), were substantially (8-fold or more) reduced in steroidogenic potency, indicating that these two modified species did have altered biological activity. Discussion
We have described conditions for the incorporation of sulfhydryl groups into protein carboxyl groups using a two step procedure. The method is related to that developed by Fattom et al. [5] for incorporation of sulfhydryl groups into acidic carbohydrates, but employs high cystamine levels during coupling. The first step is a carbodiimide mediated addition of cystamine, which contains a disulfide function. The second step is reductive generation of the free sulfhydryl group. The number of sulfhydryl groups that may be incorporated is dependent on the number of acidic side chains, but reaction conditions can be adjusted to obtain the desired degree of derivatization. This procedure was tested using carbonic anhydrase and ACTH as proteins, and derived proteins with full biological activity were obtained. A potential difficulty associated with carbodiimide mediated addition of amines such as cystamine to protein carboxyl groups is the competition of protein amino groups with cystamine amino groups for the activated carboxyl function. The outcome of protein amino group reaction is inter- and intrachain peptide bond formation. We have utilized high cystamine concentrations (1.0 M in amino groups) to limit protein to protein peptide bond formation. Given this requirement for a high concentration of cystamine amino groups, the primary variables that can be altered to affect the amount of sulfhydryl group incorporation are carbodiimide concentration and pH. As expected, both carbonic anhydrase and ACTH showed increased incorporation of sulfhydryl groups at higher concentrations of ECD. High levels of sulfhydryl group incorporation into carbonic anhydrase using this procedure did not effect
385 esterase activity of the enzyme. At pH 6.0, the level of sulfhydryl group incorporation was sharply reduced compared to that observed at pH 4.2. However, even at this pH, an average of two sulfhydryl groups were incorporated per carbonic anhydrase molecule, a quantity that is sufficient for many types of labeling reactions. It is likely that specific carboxyl groups with high pK a values could be preferentially modified by carrying out the coupling reaction at higher pH values. Sulfhydryl containing ACTH peptides were prepared that contained one sulfhydryl group per peptide molecule. Of these, two were substantially reduced in biological activity and five had activities close to that of native ACTH. The seven different modified ACTH forms obtained may represent peptides modified at the seven different carboxyl groups. It is likely that one of the peptides with substantially reduced activity was modified at Glu 5, which is known to be involved in receptor binding. That the majority of the modified ACTH peptides retain substantial activity suggests that c-amino groups of lysine residues, which are known to be involved in receptor binding, are not participating to a significant degree in inter- or intrachain peptide bond formation during the ECD mediated coupling reaction. Introduction of sulfhydryl groups into proteins lacking sulfhydryl groups in their native form permits specific reaction with a variety of commercially available crosslinking and labeling reagents. The procedure we have described for incorporation of sulfhydryl groups at protein carboxyl sites will complement previously described methods for incorporation of sulfhydryl groups at other protein sites [2,3], and may be particularly useful for proteins such as apolipoprotein [18] and luciferase [19], where lysine modification reactions are known to inhibit function.
Acknowledgements The support of the U N L Research Council and the NIH Biomedical Research Support Program and helpful discussions with Dr. Herman Knoche are gratefully acknowledged.
References 1 Torchinsky, Y.M. (1975) Sulfur in Proteins, pp. 232-236, Pergamon Press, Oxford. 2 Klotz, I.M. and Heiney, R.E. (1962) Arch. Biochem. Biophys. 96, 605-612. 3 Weston, P.D., Devries, J.A and Wrigglesworth, R. (1980) Biochim. Biophys. Acta 612, 40-49. 4 Hoare, D.G. and Koshland, D.E., Jr. (1966) J. Am. Chem. Soc. 88, 2057-2058. 5 Fattom, A., Vann, W.F., Szu, S.C., Sutton, A., Li, X., Bryla, D., Schiffman, G., Robbins, J.B. and Schneerson, R. (1988) Infect. Immun. 56, 2292-2298. 6 Laemmli, U.K. (1970) Nature 227, 680-685. 7 Buckley. D.I., Yamashiro, D. and Ramachandran. J. (1981) Endocrinology 109, 5-9. 8 Nyman, P.O. and Lindskog, S. (1964) Biochim. Biophys. Acta 85, 141-151. 9 Allen, G. (1981) Sequencing of Proteins and Peptides, pp. 26-27, Elsevier, Amsterdam. 10 Eilman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-450. 11 Duff, T.A. and Coleman, J.E. (1966) Biochemistry 5, 2009-2019. 12 Ray, P. and Strott, C.A. (1978) Endocrinology 103, 1281-1288. 13 Lowry, P.J., McMartin, C. and Peters, J. (1973) J. Endocr. 59, 43-55. 14 Boilean, G., Barbeau, C., Jeanett, L., Chretien, M. and Drouin, J. (1984) Nucleic Acids Res. 11, 8063-8071. 15 Carraway, K.L. and Koshland, D.E. (1968) Biochim. Biophys. Acta 160, 274-276. 16 Lundblad, R.L. and Noyes, C.M. (1984) Chemical Modification of Proteins, Vol. 2, pp. 105-121, CRC Press, Boca Raton, FL. 17 Fujino, M. Hatanaka, C. and Nishimura, O. (1971) Chem. Pharm. Bull. 18, 1066-1068. 18 Innerarity, T.L., Friedlander, E.J., Rail, S.C., Weisgraber, K.H. and Mahley, R.W. (1983) J. Biol. Chem. 258, 12341-12347. 19 Welches, W.R. and Baldwin, T.O. (1981) Biochemistry 20, 512-517.
Biochimica et Biophysica Acta, 1038 (1990) 382-385
Elsevier BBAPRO 33636
Introduction of sulfhydryl groups into proteins at carboxyl sites * C h a o m e i Lin, K e v i n A. Mihal * * a n d Rick J. K r u e g e r Department of Biochemistry and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE (U.S.A)
(Received 18 December1989)
Key words: Sulfbydrylgroup; Protein modification;Carbodiimide A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.
Introduction Incorporation of sulfhydryl groups into proteins is frequently employed to facilitate formation of covalent crosslinks to other proteins or labeling reagents. The variety of reagents available which react rapidly and specifically with sulfhydryl groups makes them the group of choice in many cases. While sulfhydryl group incorporation at various protein functional groups has been studied [1], derivatization of amino groups has been employed most frequently (e.g. Refs. 2 and 3). The development of water soluble carbodiimides [4] has permitted relatively selective incorporation of amines at protein carboxyl groups. Fattom et al. [5] have described a procedure for incorporating sulfhydryl groups at carboxyl sites of carbohydrates. This two-step procedure utilized ECD mediated incorporation of cystamine into N-acetylmannoseaminouronic acid to form an amide bond between the amino group of cystamine and the carboxyl group of the carbohydrate. The disulfide was then reduced to yield a carbohydrate containing a cysteamide sulfhydryl group. * Agricultural Research Division, University of Nebraska Journal Series No. 9102. * * Present address: Department of Biochemistry,Boston University School of Medicine, Boston, MA 02118, U.S.A. Abbreviations: ECD, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ACTH, adrenocorticotropichormone; EDs0, dose resulting in 50% of maximal response; SDS, sodium dodecyl sulfate. Correspondence: R.J. Krueger, Room 314, Department of Biochemistry, Universityof Nebraska-Lincoln,Lincoln,NE 68583-0718,U.S.A.
We show here that this sulfhydryl group incorporation scheme can be used to introduce sulfhydryl groups directly into proteins. The procedure was utilized to make thiolated enzymes and polypeptide hormones that retained full biological activity. Materials and Methods Materials
ECD, cystamine, dithiothreitol, bovine carbonic anhydrase, p-nitrophenyl acetate, and porcine ACTH were purchased from Sigma Chemical Co. Carbonic anhydrase was examined by SDS-polyacrylamide gel electrophoresis [6] and appeared to be better than 99% homogeneous. A C T H was purified by ion-exchange chromatography on Whatman CM-52 cellulose [7] and by preparative reversed phase HPLC on a Baker 15 # m 1 x 25 cm C18 wide pore colunm. E C D mediated incorporation of cystamine into protein
Incorporation of cystamine into proteins was carried out at 2 2 ° C in 0.5 M cystamine, 0.1 M Mes-NaOH (pH 4.2) unless otherwise noted. Carbonic anhydrase and A C T H concentrations were 0.2 mM. The reaction was initiated by the addition of ECD. The carbonic anhydrase modification reaction was 30 min in length, and the derivatized protein was separated from excess cystamine by precipitation with ammonium sulfate (added to 80% of saturation). The precipitate was collected by centrifugation and dissolved in 50 mM Tris-HC1 (pH 7.0). Dithiothreitol was added to a final concentration of 6 mM, and the sample was incubated for 20 min at room temperature to reduce the incorpo-
0167-4838/90/$03.50 © 1990 Elsevier SciencePubfishers B.V. (BiomedicalDivision)
383 rated disulfide groups to the sulfhydryl form. After reduction, the protein was precipitated with a m m o n i u m sulfate twice as described above to separate the carbonic anhydrase derivative from dithiothreitol and 2aminoethanethiol that was released from the protein by reduction. T h e derived protein was dissolved in 0.1 M Tris-HC1 and assayed as described below. The reaction conditions for sulfhydryl group incorporation into A C T H were similar to those for carbonic anhydrase except that the ECD concentration was 2 m M unless otherwise noted. Products from the incorporation of cystamine into A C T H were separated by chromatography on a Sephadex G-10 column equilibrated with 0.1 M acetic acid, and peptides obtained from this separation were reduced with dithiothreitol as above. The products were then subjected to reversed phase H P L C on a Bakerbond C18 wide pore 5 #m, 4.6 × 250 m m column using linear gradients of 0.05% trifluoroacetic acid (solvent A) and 0.05% trifluoroacetic acid, 50% acetonitrile (solvent B). The peptide fractions collected from H P L C separations were taken to dryness under vacuum, and analyzed as described below.
Analysis of modified proteins Protein concentrations of carbonic anhydrase [8] and A C T H [9] were monitored by ultraviolet absorption spectroscopy. Sulfhydryl group content of proteins was measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) [10]. Carbonic anhydrase activity was measured by a p-nitrophenyl acetate esterase assay [11], which was conducted in 0.1 M Tris-HC1 ( p H 7.4). The biological activity of A C T H was assessed by the capacity of peptides to stimulate steroidogenesis in suspensions of rat adrenocortical cells prepared as described by Ray and Strott [12]. Cell suspensions were incubated with a range of concentrations of peptide for 1 h and assayed for fluorogenic steroid content [13]. Peptide dose vs. steroidogenic response curves were plotted, and the peptide concentration yielding 50% of the maximal steroidogenic rate (EDs0) was determined. Results
The influence of carbodiimide concentration on sulfhydryl group incorporation was examined for carbonic anhydrase in the 1 to 100 m M E C D range and for A C T H in the 2.5 to 15 m M E C D range at p H 4.2. Increasing the E C D concentration caused an increase in the number of cystamine molecules incorporated per protein molecule. The sulfhydryl group content was determined after reduction of the covalently bound, cystamine derived disulfide to the sulfhydryl form (Fig. 1). The degree of sulfhydryl group incorporation is expressed as the percentage of the m a x i m u m possible, assuming the incorporation of one sulfhydryl group per carboxyl site, 32 carboxyl groups per carbonic anhydrase
E 70 E
/
60
50
/
"O
40
0
o
o
& ,30 L 0
20 i 10, I 0 0.0
I
I
I
I
0.5
1.0
1.5
2.0
log [ECDI ( m M ) ] Fig. 1. ECD concentration dependence of incorporation of sulfhydryl groups into carbonic anhydrase and ACTH. Modified carbonic anhydrase and ACTH were prepared as described in Materials and Methods. Percent maximal incorporation is based on 32 carboxyi groups per carbonic anhydrase molecule and 7 carboxyl groups per ACTH molecule, o, carbonic anhydrase; O, ACTH. molecule [8] and 7 carboxyl groups per A C T H [14]. The highest levels of sulfhydryl group incorporation observed were 22 sulfhydryl groups per carbonic anhydrase molecule at 100 m M ECD and 2.1 sulfhydryl groups per A C T H molecule at 15 m M ECD. Ultraviolet absorption spectra [9] of A C T H derivatives at this level of sulfhydryl group incorporation showed no reduction in the tyrosine to tryptophan ratio, indicating that tyrosine residues had not reacted with E C D to form O-aryl isourea adducts [15]. Because m a n y proteins are not stable at p H 4.2, the incorporation of sulfhydryl groups was determined at higher p H values with carbonic anhydrase. A striking decrease in sulfhydryl group incorporation was observed (Table I). This result was anticipated because the carboxylic acid rather than the carboxylate ion is usually considered to be the reactive species [16]. Analysis of the p-nitrophenyl acetate esterase activity of modified carbonic anhydrase showed that introduction of up TABLE I Effect of pH on sulfhydryl group incorporation into carbonic anhydrase. ECD (100 mM) mediated reaction of cystamine with carbonic anhydrase, reduction of the incorporated cystamine disulfides to free thiols, and determination of protein concentration, sulfhydryl group content, and esterase activity were carried out as described in Materials and Methods. Reactions at pH 4.2 and 5.0 were carried out in 0.1 M Mes-NaOH. The reaction at pH 6.0 was carried out in 0.1 M potassium phosphate, and the reaction at pH 7.4, which lacked ECD, was in 0.1 M Tris-HC1. pH 4.2 5.0 6.0 7.4 (no ECD)
mol -SH/mol carbonic anhydrase 22.1 8.8 2.1 0
Esterase activity (%) 87 100 98 100
384
1
E C
0 v W
o
3 4
6
a,, o t/]
I
I
I
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I
10
15
20
25
30
35
TIME (min) Fig. 2. Separation of sulfhydryl containing ACTH peptides by C18 HPLC. Incorporation of cystamine into ACTH at 2 mM ECD and separation of modified peptide from excess cystamine was carried out as described in Materials and Methods. The modified peptide was then reduced with 0.1 M dithiothreitol, and the sulfhydryl containing ACTH peptides were separated by C18 HPLC. The bars on the chromatogram indicate where fraction collection was initiated and terminated.
to nine sulflaydryl groups per molecule had very little effect on enzymatic activity, and even at 22 sulfhydryl groups per molecule, the inhibitory effect was slight
(13%). Porcine ACTH contains seven carboxyl groups. Of these only one, Glu 5, is thought to be directly involved in adrenocortical cell ACTH receptor binding associated with stimulation of steroidogenesis [17]. To obtain ACTH peptides with minimal loss of receptor binding activity, a sulfhydryl group incorporation reaction was carried out at a low ECD concentration to generate a peptide population that contained an average of 0.3 sulfhydryl groups per molecule. The object was to limit modification to at most one carboxyl group per ACTH molecule. Peptide products were then reduced to their sulfhydryl form and resolved by C18 HPLC (Fig. 2). TABLE II Sulfbydryl group content and steroidogenic potency of modified ACTH species. Preparation of sulfhydryl containing ACTH peptides, purification, and their analysis are described in Materials and Methods. The fraction numbers refer to the peaks indicated in Fig. 2. The EDs0 associated with peak 1 was equal to that of native ACTH. Fraction No.
mol -SH/mol ACTH
EDs0 (nM)
1 2 3 4 5 6 7 8
0.02 0.69 0.98 1.06 1.01 1.15 0.94 0.32
0.16 0.25 0.13 0.19 0.13 0.16 1.26 3.97
The peptides eluting at and after the unmodified form of ACTH (peak 1) were collected and analyzed for peptide and sulfhydryl content as well as capacity to stimulate steroidogenesis in suspensions of rat adrenocortical cells. Seven modified peptide fractions were obtained. The yield of sulflaydryl containing peptide (peaks 2 through 8) was 25% of the total peptide eluted from the C18 column. Five of these fractions (peaks 3-7) contained approximately one sulfhydryl group per peptide molecule, while non-integer values were obtained for peaks 2 and 8 (Table II). Peptide peaks 2 through 8 all stimulated steroidogenesis to the same maximal level observed with native ACTH. However, while dose-response analyses showed that peaks 2 to 6 had EDs0 values close to that of native ACTH, two of the peaks (7 and 8), were substantially (8-fold or more) reduced in steroidogenic potency, indicating that these two modified species did have altered biological activity. Discussion
We have described conditions for the incorporation of sulfhydryl groups into protein carboxyl groups using a two step procedure. The method is related to that developed by Fattom et al. [5] for incorporation of sulfhydryl groups into acidic carbohydrates, but employs high cystamine levels during coupling. The first step is a carbodiimide mediated addition of cystamine, which contains a disulfide function. The second step is reductive generation of the free sulfhydryl group. The number of sulfhydryl groups that may be incorporated is dependent on the number of acidic side chains, but reaction conditions can be adjusted to obtain the desired degree of derivatization. This procedure was tested using carbonic anhydrase and ACTH as proteins, and derived proteins with full biological activity were obtained. A potential difficulty associated with carbodiimide mediated addition of amines such as cystamine to protein carboxyl groups is the competition of protein amino groups with cystamine amino groups for the activated carboxyl function. The outcome of protein amino group reaction is inter- and intrachain peptide bond formation. We have utilized high cystamine concentrations (1.0 M in amino groups) to limit protein to protein peptide bond formation. Given this requirement for a high concentration of cystamine amino groups, the primary variables that can be altered to affect the amount of sulfhydryl group incorporation are carbodiimide concentration and pH. As expected, both carbonic anhydrase and ACTH showed increased incorporation of sulfhydryl groups at higher concentrations of ECD. High levels of sulfhydryl group incorporation into carbonic anhydrase using this procedure did not effect
385 esterase activity of the enzyme. At pH 6.0, the level of sulfhydryl group incorporation was sharply reduced compared to that observed at pH 4.2. However, even at this pH, an average of two sulfhydryl groups were incorporated per carbonic anhydrase molecule, a quantity that is sufficient for many types of labeling reactions. It is likely that specific carboxyl groups with high pK a values could be preferentially modified by carrying out the coupling reaction at higher pH values. Sulfhydryl containing ACTH peptides were prepared that contained one sulfhydryl group per peptide molecule. Of these, two were substantially reduced in biological activity and five had activities close to that of native ACTH. The seven different modified ACTH forms obtained may represent peptides modified at the seven different carboxyl groups. It is likely that one of the peptides with substantially reduced activity was modified at Glu 5, which is known to be involved in receptor binding. That the majority of the modified ACTH peptides retain substantial activity suggests that c-amino groups of lysine residues, which are known to be involved in receptor binding, are not participating to a significant degree in inter- or intrachain peptide bond formation during the ECD mediated coupling reaction. Introduction of sulfhydryl groups into proteins lacking sulfhydryl groups in their native form permits specific reaction with a variety of commercially available crosslinking and labeling reagents. The procedure we have described for incorporation of sulfhydryl groups at protein carboxyl sites will complement previously described methods for incorporation of sulfhydryl groups at other protein sites [2,3], and may be particularly useful for proteins such as apolipoprotein [18] and luciferase [19], where lysine modification reactions are known to inhibit function.
Acknowledgements The support of the U N L Research Council and the NIH Biomedical Research Support Program and helpful discussions with Dr. Herman Knoche are gratefully acknowledged.
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