An immunochemical aid to sequence determination of proteins

ANALYTICAL

BIOCHEMISTRY

80,

10%

An lmmunochemical ANTHONY Department

J. BRAKE,

115

(1977)

Aid to Sequence of Proteins1

Determination

FRANCO CELADA~, AUDFU?E V. FOWLER, IRVING ZABIN

of Biological Chemistry, School of Medicine, University of California, Los Angeles,

and Molecular Biology California 90024

AND Institute,

Received December 6, 1976: accepted January 11, 1977 A specific radioimmunoassay for peptides has been developed using *Z51-Iabefed peptides and a double-antibody precipitation. Cross-reacting peptides are measured by inhibition of the binding of the labeled cyanogen bromide peptide to its antibody. The assay, which allows detection of picomole quantities, was used to monitor the purification of two overlapping tryptic peptides from a complex mixture of peptides. These were shown to contain a portion of the sequence of the radio-labeled cyanogen bromide peptide and a portion of the sequence of a cyanogen bromide peptide which follows in the polypeptide chain. The need to analyze many fractions in a digest in order to locate a desired peptide is thus avoided. The general suitability of this method for the purification of specific peptides from digestion mixtures of other large proteins is discussed.

Radioimmunoassays have provided extremely sensitive and specific methods for the measurement of peptide hormones and other proteins. These radioassays have also been used to provide means of detection of selected portions of a peptide chain. For example, antisera have been prepared against fragments of parathyroid hormone produced by cleavage of the hormone or by synthesis (1). In this paper, we present an extension of the radioimmunoassay procedure to aid in the amino acid sequence determination of proteins. The primary structure of&galactosidase fromEscherichia cofi has been under study in this laboratory for some time (2-7). This very large protein, which contains more than 1000 amino acid residues in a single polypeptide chain, yields 24 peptides upon cleavage with cyanogen bromide. The separation, sequence determination, and ordering of these peptides will be described elsewhere. During the course of this work, antibodies have been prepared against a number of the cyanogen bromide peptides for the purpose ofprobing the conformation of the native protein (F. Celada, A. V. Fowler, and I. Zabin, in preparation). It occurred to us that some of these 1 This work was supported, in part, by Grants AI 04181 and 07104 from the U. S. Public Health Service. 2 Recipient of a fellowship from The Italo-American Medical Education Foundation. Present address: Laboratorio di Biologia Cellulare, Via Romagnosi 18, Roma 001% Italy. 108 Copyright All rights

Q 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN ooO3-2697

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OF RIA

IN PROTEIN

SEQUENCING

109

antibodies might also be useful for another purpose, to help monitor the isolation of overlapping peptides necessary for the correct ordering of peptides in the polypeptide chain. An example of the successful application of such a procedure is described here. MATERIALS

AND METHODS

Materials. p-Galactosidase was obtained as previously described (8). Carrier-free sodium [1251]iodide was from New England Nuclear. Goat anti-rabbit immunoglobulin antiserum (Catalog No. 539844, 1 unit precipitates 40 pg of rabbit y-globulin) and lactoperoxidase (B grade) were purchased from Calbiochem, and complete Freund’s adjuvant from Difco. Chromatography materials used were Sephadex G-50 and SP-Sephadex C-50 (Pharmacia), Bio-Gel P-2 (Bio-Rad), and CM-cellulose (CM52, Whatman). Preparation and sequence analysis of peptides. The cyanogen bromide peptides of /3-galactosidase, CB21 and CB22, were purified and sequenced by methods described elsewhere (5-7). Tryptic peptides were prepared from 1.3 g of carboxymethyl /?-galactosidase which was treated with a 20-fold excess of citraconic anhydride (9). Digestion was carried out at 22°C with a trypsin:substrate ratio, by weight, of 1:200. After 2.5 hr an equal amount of trypsin was added, and incubation was continued for 1 hr. The peptide mixture in 30% acetic acid was passed through a column of Sephadex G-25 (5.0 x 150 cm), and the peptides excluded from the gel were collected and made to a volume of 100 ml of 5% acetic acid. These were then extracted four times with equal volumes of n-butanol. The butanol extracts were combined and dried in vacua, yielding 0.32 g of peptides. Antibody to CB21. CB21, 125 pg (18 nmol) in 0.25 ml of 0.1 M sodium phosphate, pH 7.0, was homogenized in an equal volume of complete Freund’s adjuvant and injected into the footpads of New Zealand white rabbits. An identical injection was made 30 days later. Fifteen days after the second injection, an additional 10 pg of peptide dissolved in the same buffer was injected intradermally in several locations on the back. Antisera were collected 7 days after the last injection. The antiserum used was estimated from inhibition curves to have a binding capacity of 12 nmol of CB2llml of undiluted serum. Radioiodination of CB21. CB21, 25 pg (3.6 nmol) in 0.05 ml of 0.1 M NaH,PO,, 0.02% sodium azide, pH 7.0 (Buffer A), was added to a mixture of 300 @i of sodium [1251]iodide in 50 ~1 of 0.5 M KH2P04, pH 7.5, and 5 ~1 of 1 mg/ml of lactoperoxidase. Hydrogen peroxide, 0.0015% in 0.05 M KH2P04, pH 7.5, was added in fourS-pl aliquots at intervals of 3 min. Three minutes after the last addition, the reaction was stopped by the addition of 0.1 ml of KI, 10 mg/ml in 0.05 M KH2POI, pH 7.5. The solution was then

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BRAKE ET AL.

FIG. 1. Amino acid sequences of cyanogen bromide peptides CB21 and CB22 and tryptic peptides 1A and IB.

applied to a column of Bio-Gel P-2 (0.9 x SOcm) equilibrated with Buffer A and was washed into the column with 0.2 ml of the KI solution. The column was eluted with Buffer A. The fractions corresponding to the first peak of radioactivity were pooled, and bovine serum albumin was added to a concentration of 1 mg/ml. The [1251]CB21 had a specific radioactivity of approximately 2 x 10’ cpm/nmol. Aliquots of this solution were stored at -40°C. Radioimmunoassay. The radioimmunoassay described here is modified from that described by Celada et al. (10). Peptides to be tested for inhibition, which were normally stored in 30% acetic acid, were dried with 100 ,cLgof albumin in 6 x 50 mm test tubes in a vacuum oven below 50°C. They were then incubated in a total volume of 200 ~1 of Buffer A, containing 0.25% Triton X-100 and 50 ~1 of a 1:50 dilution of anti-CB21.

30

OO

IO

50

Unlabclled

CBZI,

pmolcs

100

FIG. 2. Calibration curve for radioimmunoassay of CB21. Varying amounts (in duplicate) of unlabeled CB21 were preincubated with rabbit antiserum before the addition of [‘251]CB21 (30,000 cpm) to each sample. The amount of radioactivity was measured in the precipitate formed by the addition of goat anti-rabbit immunoglobulin.

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USE OF RIA IN PROTEIN SEQUENCING

0 .6s I”

0 ‘.4 -

I

0

20

60

100 FRACTION

140

160

NUMBER

FIG. 3. Sephadex G-50 chromatography of tryptic peptides. The fraction unabsorbed on CM-cellulose was applied to a column of Sephadex G-50 (2.5 x 200 cm) in 30% acetic acid. After elution of 255 ml, 3.4-m] fractions were collected, and the absorbance at 280 nm was monitored (solid line). Fractions were pooled as indicated by the solid horizontal bars and were evaporated to volumes of 2 ml. Duplicate 0.05~~1 portions of each pooled fraction were used in the CB21 radioimmunoassay. The results were expressed as immunoequivalents of CB21 (vertical bars).

After 30 min, 25 ~1 of [1251]CB21 (4 pmol; 30,000-90,000 cpm) was added, and the mixture was incubated at room temperature for 1 hr. Goat anti-rabbit immunoglobulin, 20 ~1, 25 unit/ml, was then added, and the tubes were kept at 4°C overnight. The tubes were then centrifuged, and the supernatant solution was removed by aspiration. The precipitates were washed with 0.6 ml of Buffer A containing 0.25% Triton X-100 and were centrifuged again. The radioactivity present in the precipitates was determined in a Beckman Biogamma counter. RESULTS

The amino acid sequences of two cyanogen bromide peptides from /3-galactosidase, numbered CB21 and CB22, are shown in Fig. 1. From evidence obtained during work on the primary structure, it seemed likely that CB22 followed CB21 in the polypeptide chain. However, direct proof, by isolation of an overlapping peptide, was lacking. A tryptic peptide could supply the missing evidence, but no such peptide had been obtained from tryptic digests. While working on a tryptic digest of /3-galactosidase which had been pretreated with citraconic anhydride to block hydrolysis at lysine residues, it was noted that the extent of cleavage at each arginine was not complete, resulting in the production of many unexpectedly large peptides.

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One such large peptide, if it contained most of the sequence of CB21, would be expected to be immunologically similar to CB21 and should cross-react with anti-CB2 1, This turned out to be the case. Antibody to CB21 was first prepared and tested by a binding assay against lz51-labeled CB21, as shown in Fig. 2. When unlabeled CB21 in the range of l-300 pmol was added as inhibitor, the amount of binding of label was decreased. Other cyanogen bromide peptides, even at concentrations of 100 pmol, did not inhibit binding. When nonimmune sera replaced the antisera in the assay, less than 0.5% of the total counts were found in the precipitate. This assay was then used to test for the presence of a cross-reacting peptide. A tryptic digest of citraconyl-P-galactosidase was extracted with butanol to obtain the bulk of the larger peptides. When a quantity of the butanol-soluble fraction corresponding to about 100 pmol of peptides, based on amino acid analysis, was added, an amount of inhibition equivalent to that found with 80 pmol of CB21 was observed. Therefore, this fraction apparently contained a tryptic peptide or peptides with part of the amino acid sequence in CB21. The peptide mixture in 0.02 M ammonium acetate, 8 M urea, pH 5.0, was applied to a carboxymethyl cellulose column (5), and the pooled fractions were tested for cross-reactivity. The material which was not absorbed on the column contained the major amount of inhibitory material. After passage through Sephadex G-50 (Fig. 3), two peaks, 1A and lB, were found

FIG. 4. Cross-reactivity of purified 1A and 1B with CB21. Parallel radioimmunoassays were done with the three unlabeled peptides in competition with 60,000 cpm of [1251]CB21. (0) CB21, (m) IA, (A) 1B.

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USE OF RIA IN PROTEIN SEQUENCING TABLE AMINO

ACID

Amino acid Tryptophan Lysine Histidine Arginine Carboxymethyl Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Vahne Methionine Isoleucine Leucine Tyrosine Phenylalanine

cysteine

COMPOSITION

1 OF TRYPTIC

PEPTIDES

IB - (2)” 0.08 2.89 (3) 0.87 (I) 6.37 (7) 1.83 (2) 1.66 (2) 3.23 (3) 3.76 (4) 3.02 (3) 2.23 (2) 1.99 (2) 0.97 (1) 0.35 5.22 (6) 2.44 (3) 1.93 (2)

1A

-

(2)

3.40 (4) 1.97 (2) 7.95 (8) 1.95 (2) 2.14 (2) 6.44 (6) 4.46 (4) 4.72 (4) 4.31 (4) 2.99 (3) 0.93 (1) 0.95 (1) 7.52 (8) 2.61 (3) 1.84 (2)

(1Integral number based on sequence analysis and known sequence of CB21.

to be the most active. Each material was then chromatographed on SP-Sephadex and was finally passed again through Sephadex G-50. The two cross-reacting peptides, 1A and IB, were tested in the radioimmunoassay in parallel with unlabeled CB21. The three curves are very similar, with the two peptides showing maximal inhibition, more than 90% of that produced by CB21 (Fig. 4). The amino terminal residues of peptides 1A and 1B were found to be isoleucine and valine, respectively. Upon treatment with cyanogen bromide, each peptide yielded tyrosine as an additional amino-terminal amino acid. The amino acid compositions of the two peptides are shown in Table I. Automatic sequence analysis to residue 42 of peptide 1B showed that it corresponded to a fragment of 44 residues containing the last 31 residues of CB21 and the first 13 residues of CB22. Comparison of the amino acid compositions and consideration of the amino-terminal data indicate that peptide 1A differs from 1B only by the presence of an additional 13 aminoacid residues at its NH,-terminal end. The sequences of the two overlapping peptides are shown in Fig. I. DISCUSSION

We have described a highly sensitive radioimmunoassay for peptides. The method depends on the binding of ‘251-labeled antigen to its antibody

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and precipitation of the complex with anti-antibody. Cross-reacting peptides are measured by inhibition of binding. Picomole quantities (usually less than 1 pg) can be detected easily. One of the keys to the success of the procedure is the inclusion in the buffer solution of Triton X-100, found to be necessary to reduce nonspecific binding and to maintain the solubility of some peptides. The experiments described here were carried out with antibody prepared against a 61-residue cyanogen bromide peptide. Immune seraagainst many other /3-galactosidase peptides, of 23 or more amino acid residues, have been successfully prepared without the need for coupling to a protein carrier (F. Celada, A. V. Fowler, and I. Zabin, in preparation). The cyanogen bromide peptide used in this example (CB21) was iodinated with lactoperoxidase. Peptides lacking tyrosine but containing histidine have been iodinated by the chloramine T method (11). Use of other labeling techniques might allow the radioimmunoassay of peptides lacking both tyrosine and histidine. With [1251]CB21 and anti-CB21, two related cross-reacting tryptic peptides were easily detected in complex digests. Their purification could be monitored quickly and efficiently, and the need to analyze many fractions by more time-consuming analytical methods was avoided. Analysis of the two peptides so purified (IB and 1A) revealed that they were overlapping peptides containing 3 1 and 44 residues, respectively, from the carboxyl-terminal portion of CB21 and 13 residues from the amino-terminal end of CB22. The nearly identical antigenic behavior of CB21 and peptides 1A and 1B in this assay are of interest in defining the antigenic nature of CB21. The results suggest that only the carboxyl-terminal 31 residues of CB21 are responsible for its antigenicity in this case. Thus, use of other overlapping peptides and fragments from other peptides might be useful in defining antigenic determinants in other antigens. The approach used here should be of general use in identifying overlapping peptides among the cleavage products of other large proteins. Such methods would be most useful in the final stages of protein sequence determination when only a few overlapping peptides are needed. Radioassays for specific peptides might also be useful in purifying altered peptides from mutant strains. ACKNOWLEDGMENTS We wish to acknowledge the expert technical assistance of Val H. Schaeffer and Paulette Osborne.

REFERENCES 1. Seyre, G. V., Tregear, G. W., and Potts, J. T. (1975) in Methods in Enzymology (O’Malley, B. W., and Hardman, J. G., eds.), Vol. 37, Part B, pp. 38-66, Academic Press, New York.

USE OF RIA IN PROTEIN 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

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Fowler, A. V., and Zabin, I. (1970) J. Biol. Chem. 245, 5032-5041. Fowler, A. V. (1972) J. Biol. Chem. 247, 5425-5431. Zabin, I., and Fowler, A. V. (1972) J. Biol. Chem. 247, 5432-5435. Fowler, A. V. (1975) in Solid Phase Methods in Protein Sequence Analysis (Laursen, R. A., ed.), pp. 169-177, Pierce Chemical Company, Rockford, Illinois. Langley, K. E., Fowler, A. V.. and Zabin, I. (1975) J. Biol. Chem. 250, 2587-2592. Langley, K. E.. Villarejo, M. R., Fowler, A. V., Zamenhof, P. J., and Zabin, I (1975) Proc. Nat. Acad. Sci. USA 72, 1254-1257. Fowler, A. V. (1972) J. Bacteriol. 112, 856-860. Atassi, M. Z., and Habeeb, A.F.S.A. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.). Vol. 25, pp. 546-553, Academic Press, New York. Celada, F., Natali, P. G., and Radojkovic, J. (1976) J. Zmmunol. 117, 904-910. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123.