THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 24, Issue of December 25, pp. 15572-15578,1984 Printed in U. S. A .

A General Procedure for the End Labeling of Proteins and Positioning of Amino Acids in the Sequence*

(Received for publication, July 2, 1984)

Daniel G. Jay From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

This paper reports a procedure for the specific ra- diolabeling of the amino termini of proteins. By Edman degradation, a protein is protected at all lysine amino groups while retaining a free amino terminus and such a modified protein is end-labeled by an amino group- specific reagent (radioiodinated Boiton-Hunter re- agent). Partial proteolyses with a variety of specific amino acid cleaving reagents generate a series of frag- ments which predict the location of the specific amino acids in the primary structure. The amino acids deter- mined so far include Arg, Asp, Cys, Glu, Met, Trp, and Asn-Gly. The procedure is demonstrated on 8-galacto- sidase and X immunity 434 repressor protein. One of the uses of the procedure, the identification and local- ization of point mutations within the sequence, is illus- trated using X immunity 434 repressor protein.

DNA sequencing (Maxam and Gilbert, 1980; Sanger et al., 1977) has led to significant advances in molecular biology. This technique was made possible by the ability to: 1) label DNA at only one end; 2) resolve fragments of DNA by size differing by a single nucleotide; and 3) cleave specifically at each of the four bases. An analogous procedure for protein sequencing would also be quite useful; however, none of these three requirements exists for proteins. Although there exist reagents that react specifically with amino groups or carboxyl groups, the existence of these groups on amino acid residues in the protein would result in internal labeling at these sites.

SDS’-polyacrylamide gel electrophoresis can be used to separate proteins on the basis of size (Weber and Osborn, 1969), but the resolution is limited by the heterogeneity of peptides and slight differences in SDS binding. Thus, the separation of protein fragments differing by a single amino acid residue has not yet been achieved.

There exist several procedures for the cleavage of proteins a t specific amino acids. There are specific proteases for lysine and arginine (Smyth, 1967; Schenkein et al., 1977), glutamate and aspartate (Drapeau et al., 19721, and proline (Yoshimoto et al,, 1980). Also, there are chemical cleavage reagents specific for methionine (Gross, 1967), cysteine (Jacobson et al., 1973), tryptophan (Schecter et al., 1976), aspartate (Tsung and Fraenkel-Conrat, 1965), tyrosine (Wilchek and Witkop, 1967), and the dipeptides Asp-Pro (Landon, 1977) and Asn-

* This research was supported by Grant HL 08893 from the Na- tional Institutes of Health to G. Guidotti, Harvard University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PITC, phenylisothiocyanate; DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid).

Gly (Bornstein, 1970). Knowledge of the positions of these residues in the primary structure of a protein would be useful in protein structure studies.

Although sequencing is not possible for the reasons cited, a procedure analogous to DNA sequencing would be useful for positioning specific amino acids in the sequence. Such a partial assignment would be useful as a means of checking protein or DNA sequencing. This procedure would signifi- cantly increase the amount of information obtainable from peptide maps of the products of partial proteolysis since the positions of amino acids could be determined with respect to the amino terminus. By comparing similar proteins, not only could differences be demonstrated, but the location of these differences could be mapped. In short, even with the limita- tions of resolution in gel electrophoresis and the inability to specifically cleave proteins at all 20 amino acid residues, a procedure, analogous to DNA sequencing, for the analysis of proteins possessing a specific end label would be useful in studying protein structure.

This paper reports a general procedure for the specific radiolabeling of the amino termini of proteins. This was done by reacting the product of one round of Edman degradation with an amino group-specific radiolabeling reagent. The Ed- man degradation product has all lysyl amino groups blocked and only the NH2-terminal amino group present. By perform- ing partial proteolysis with reagents of known specificity followed by analysis using SDS-PAGE and autoradiography, the approximate position of specific amino acids in the se- quence was determined by a method analogous to DNA se- quencing. By using this procedure, a point mutation in the sequence of a site-specific mutant of h immunity 434 repressor protein was identified and approximately located.

MATERIALS AND METHODS

Dansyl chloride, phenylisothiocyanate, trifluoroacetic acid, Staph- ylococcus aureus protease, and polyamide TLC plates were purchased from Pierce. Chymotrypsin, trypsin, P-galactosidase, human trans- ferrin, cyanogen bromide, dithionitrobenzoic acid, N-chlorosuccinim- ide, and hydroxylamine hydrochloride were purchased from Sigma. Wild-type and mutant X immunity 434 repressor protein were the generous gifts of R. P. Wharton (Harvard University, Department of Biochemistry and Molecular Biology). ‘251-labeled Bolton-Hunter re- agent was purchased from New England Nuclear.

Amino Acid Protection (PITCITrifluoroacetic Acid Treatment)

Protection was done by a variation of the procedure of Weiner et al. (1972). Fifty @g of protein were incubated in 100 p1 of 0.5 M NaHC03, pH 9.8, 2% SDS in a 1.5-ml Eppendorf tube at 50 “C to which were added 10 pl of phenyl isothiocyanate. The tube was immediately flushed with nitrogen, closed, and incubated for 1 h with occasional mixing. The protein was precipitated by the addition of I ml of acetone with mixing at room temperature and collected by centrifugation for 30 s in a Beckman Microfuge. The precipitate was washed five times with 0.5 ml of acetone and lyophilized. The precip- itate was dissolved in 100 pl of trifluoroacetic acid and the tube was

15572

End Labeling of Proteins 15573

flushed with nitrogen, closed, and held at 50 "C for 5 min with occasional mixing. One hundred pl of 1% SDS were added and the sample was dialyzed overnight in 1% SDS, 100 mM sodium borate, pH 8.5. Alternatively, a small amount (approximately 1 mg) of SDS was added to the samples and the samples in trifluoroacetic acid were divided into 10-pl aliquots and lyophilized.

Radiolabeling of the PZTC/Trifluoroacetic Acid-treated Protein The protein sample (10 pl) at approximately 1 mg/ml in 100 mM

sodium borate, pH 8.5, 1% SDS was labeled with Iz5I-labeled Bolton- Hunter reagent (Bolton and Hunter, 1973) a t 4 "C overnight or for 3 h a t room temperature. Radiolabeled samples were either used directly or passed over a desalting column equilibrated in 5 mM sodium phosphate, pH 8.0, 0.1% SDS, 0.25% gelatin.

Analysis of the PITC/Trifluoroacetic Acid-treated Protein

Dansylation and subsequent NHz-terminal analysis was done by the methods of Gray (1972). Peptide mapping of radiolabeled samples was adapted from the method of Markowitz and Marchesi (1981).

Specific Amino Acid Cleavages

In order to produce relatively random cleavages at a low frequency, reactions were done under denaturing conditions, a t high protein concentrations. Sample volume was minimized for better resolution in gel electrophoresis. Incubation time was dependent on the protein sample and was empirically determined. Individual incubation times are given in the appropriate figure legend.

Trypsin-Samples were incubated at 37 'C in 0.1% SDS, 10 mM sodium borate, pH 8.5, 100 pg/ml trypsin with a protease to substrate ratio of 10:l. The reaction was terminated by boiling the sample in Laemmli sample buffer for 3 min.

S. aureus Proteas-Samples were incubated a t 37 "C in 0.1% SDS, 10 mM sodium borate, pH 8.5, 100 pg/ml S. aureus protease with a protease to substrate ratio of 1O:l. The reaction was terminated by boiling the sample in Laemmli sample buffer for 3 min.

Cyanogen Bromide-Samples were reacted in 5 mg/ml cyanogen bromide in 50 mM HCl at 37 "C. After the reaction, an equal volume of 2~ Laemmli sample buffer was added and the sample was analyzed

DTNBIKCN-Samples were made 2.5 mM in dithionitrobenzoic acid, 200 mM Tris/acetate, pH 7.8, 5 mM EDTA, 1% SDS by adding an equal volume of a freshly prepared 2~ stock solution and incubated at room temperature for 10 min. KCN was added to a final concen- tration of 1 mM and the sample was kept for an additional 10 min at room temperature. One-fifth volume of 1 M Tris-HC1, pH 9, was added and the mixture was incubated at 37 "C. After the reaction, an equal volume of 2X Laemmli sample buffer was added and the sample was analyzed by SDS-PAGE.

Hydroxylamine-A 2~ stock solution was freshly prepared on ice by dissolving 0.34 g of hydroxylamine hydrochloride in 1.3 ml of water to which was added 0.25 ml of 50% NaOH, 0.5 ml of 1 M K&O, and the pH was adjusted to 10.5. To initiate the reaction, an equal volume of the hydroxylamine solution was added to the sample and the reaction was conducted a t 37 "C.

N-Chlorosuccinimide-Samples were reacted in 0.5 mg/ml N-chlo- rosuccinimide, 50 mM acetic acid, at room temperature. After the reaction, an equal volume of 2X Laemmli sample buffer was added and the sample was analyzed by SDS-PAGE.

by SDS-PAGE.

Peptide Mapping of End-labeled Protein after Partial Proteolysis

After partial cleavage, the protein samples were analyzed by SDS- polyacrylamide gel electrophoresis (Laemmli, 1970) and fragments were visualized by autoradiography for radioiodinated samples. Silver staining was done by the method of Wray et al. (1981).

RESULTS AND DISCUSSION

Method of End Labeling of Proteins-The major difficulty in specifically end labeling a peptide is the likelihood of internal labeling at residues with similar functional groups. In order to label only the end group, it is necessary to protect all similar functional groups on the protein while retaining a free end group. This was done by a novel application of the Edman degradation as outlined in Fig. 1. Phenylisothiocya- nate reacts with all amino groups. Incubation under acidic

NH - 2

1 PTC PTC

PTC - 1 TFA

PTC PTC

NH 2 - + PTH-aa

1 F2"11 - PTC. PTC

L -1

FIG. 1. General scheme for the end labeling of proteins. NH,, amino terminus; Lys, lysine residue; TFA, trifluoroacetic acid; PTC, phenylthiocarbamyl group; PTH-aa, amino acid phenylthiohydan- toin; BH, 1Z51-labeled Bolton-Hunter reagent.

conditions leads to cyclization and cleavage of the phenyl- thiocarbamyl amino acid at the amino terminus, producing a protein molecule in which all the lysyl amino groups are covalently modified and the amino terminus is free. The t-

phenylthiocarbamyl group of the modified lysine residue is not susceptible to cyclization and is stable to acidic conditions (Konigsberg, 1967). Since the resulting peptide has only the one free amino group, radiolabeling with an amino group- specific reagent such as '251-labeled Bolton-Hunter reagent will produce a protein labeled exclusively at the amino ter- minus.

Although the reactions are done on 50 pg of protein, there is no reason why they could not be carried out with submicro- gram quantities of protein. With small amounts of protein, the use of a succinylated carrier protein during the acetone precipitation step is recommended to ensure quantitative precipitation (Weiner et al., 1972).

Analysis of PITCjTrifluoroacetic Acid-treated p-Galactosid- me-@-Galactosidase was chosen as a model protein because its sequence is known, it resolves sharply on SDS-PAGE, it possesses a free amino terminus, and is easily available in large quantities.

It was necessary to demonstrate that PITC/trifluoroacetic acid treatment produced a protein with a single free amino group and that this amino group is located at the amino terminus. To show this, unreacted @-galactosidase and PITCj trifluoroacetic acid-treated @-galactosidase were reacted with dansyl chloride and acid-hydrolyzed. The hydrolysates were dried and extracted with ethyl acetate to remove most of the free dansylic acid. The samples were analyzed by two-dimen- sional TLC on polyamide plates. The results for this experi- ment are shown in Fig. 2. The positions of the spots corre- spond to those previously observed (Gray, 1972) and were also checked against prepared dansylated standards. The fluores- cent emissions of the spots corresponded to their character- istic colors; blue for dansyl hydroxide and bright yellow for

15574 End Labeling of Proteins

A B

FIG. 2. Dansylation and acid hydrolysis of untreated and PITC/trifluoroacetic acid-treated 8-galactosidase. Proteins were dansylated, acid-hydrolyzed, and analyzed by TLC on polyamide plates: in the vertical direction with solvent I, 1.5% formic acid and in the horizontal direction with solvent 11, 90% benzene, 10% acetic acid. The spots were visualized by UV fluorescence. The identity of the spots has been determined by comparison with standards. The identities of the spots are as follows: Tyr, 0-dansyl tyrosine; Lys, dansyl lysine; NH2, dansyl amide; DA, dansyl hydroxide; Thr, dansyl threonine; Met, dansyl methionine. A, untreated 8-galactosidase; E, PlTC/trifluoroacetic acid-treated 8-galactosidase.

dansyl tyrosine (Gray, 1972). In Fig. 2 A , the pattern obtained from dansylated @-galactosidase is shown. There are promi- nent spots corresponding to dansyl lysine, dansyl tyrosine, dansyl hydroxide, and dansyl amide and a fainter spot corre- sponding to the first amino acid in the sequence, dansyl threonine (Fowler and Zabin, 1977). Analysis of the dansy- lated PITC/trifluoroacetic acid-treated @-galactosidase (Fig. 2B) showed prominent spots for dansyl tyrosine, dansyl hy- droxide, and dansyl amide, but no apparent spot for dansyl lysine or dansyl threonine. One new faint spot appeared corresponding to dansyl methionine, the second amino acid in the sequence of @-galactosidase. The data show that in both cases protein was labeled by dansyl chloride as evidenced by the presence of dansyl tyrosine, but lysine appeared to be dansylated only in the untreated @-galactosidase and not in the PITC/trifluoroacetic acid-treated protein. Also, the NH2- terminal amino acid changes from the first to the second amino acid of the sequence. The simplest interpretation of these data is that the PITC/trifluoroacetic acid treatment has covalently modified all the lysine amino groups, protecting them from dansylation, and removed the first modified amino acid, thus exposing the second amino acid as a free amino terminus. Therefore, the PITC/trifluoroacetic acid-treated @- galactosidase possesses only one free amino group and the location of that amino group is at the amino terminus.

Similar analysis of untreated and PITC/trifluoroacetic acid-treated human transferrin gave a similar result (data not shown). This, coupled with the analysis of dansyl @-galacto- sidase, implies that the PITC/trifluoroacetic acid treatment is a general procedure for protecting all lysine amino groups while retaining a free amino terminus. The absence of other dansyl amino acid spots suggests the lack of internal nicking during the cyclization/cleavage reaction.

Quantitation of Labeling of Untreated, PITC-treated, and PITCITrifluoroacetic Acid-treated @-Galactosidase-The amount of label incorporated into protein by '251-labeled Bol- ton-Hunter treatment of untreated, PITC-treated, and PITC/ trifluoroacetic acid-treated @-galactosidase was compared by analyzing serial dilutions of labeled protein on SDS-PAGE and visualizing the protein by silver staining. The bands from lanes of the same silver staining intensity were removed and the radioactivity in the bands was determined. In this way, an approximate value for the ratio of label incorporated by the three species could be obtained. Untreated @-galactosidase

was shown to incorporate 50-fold more label than PITC- treated protein and 6-fold more than PITC/trifluoroacetic acid-treated @-galactosidase. This implies that very little label is incorporated into PITC-treated @-galactosidase when la- beling with an amino group-specific reagent and that labeling is substantially increased @-fold) by treatment with trifluo- roacetic acid. This is consistent with the view that PITC reacts with all the amino groups and trifluoroacetic acid treatment leads to the cyclization and cleavage of the end group, freeing the amino terminus.

These ratios are lower limit values since the amount of protein for the three samples was based on equal silver stain- ing intensities. Dion and Pomenti (1983) have shown that silver stain intensity is directly related to the mole percentage lysine composition; thus, PITC protection of lysines would result in a decrease of silver stain and an underestimate of protein for the PITC and PITC/trifluoroacetic acid-treated samples.

It should be noted that there is only a 6-fold difference in the amount of label incorporated into the FITC/trifluoroa- cetic acid-treated @-galactosidase as compared to the un- treated @-galactosidase while there are 20 more potential labeling sites (there are 20 lysines in @-galactosidase (Fowler and Zabin, 1977)). Since the labeling reaction is not quanti- tative and only a small fraction of the NH, groups is labeled by Bolton-Hunter reagent, it is not possible to predict a ratio of incorporation between the NH2 terminus and the t-NH, groups.

Peptide Mapping Analysis of Radiolabeled, Untreated, and PITC/Trifluoroacetic Acid-treated @-Galactosidase-Radiola- beled &galactosidase and PITC/trifluoroacetic acid-treated @-galactosidase were subjected to complete digestion with chymotrypsin and analyzed by two-dimensional peptide map- ping. The results are shown in Fig. 3. For untreated @- galactosidase (Fig. 3A), many spots are observed, while for the @-galactosidase radiolabeled after PITC/trifluoroacetic acid treatment one spot is predominantly observed as indi- cated by the arrow (Fig. 3B). A radiolabeled spot above this one runs at the solvent front and may correspond to free "'1 that has dissociated from the peptides during the high voltage

A B

FIG. 3. Two-dimensional peptide map of complete chymo- trypsin digestion of radiolabeled untreated and PITC/trifluo- roacetic acid-treated 8-galactosidase. After separating free Bol- ton-Hunter reagent by desalting, radiolabeled samples of 8-galacto- sidase and &galactosidase pretreated with PITC/trifluoroacetic acid were digested in 200 pg/ml chymotrypsin in 5 mM sodium phosphate, pH 8,0.1% SDS for 18 h at 37 "C. Samples were spotted on cellulose- coated TLC plates saturated with 1.5% acetic acid, 0.5% formic acid and electrophoresis was performed at 1000 V for 1.5 h. After drying the plate, thin layer chromatography was run in the second dimension in 1-butano1:pyridine:acetic acid:water (32.5:25:5:20) and the spots were visualized by autoradiography. A, 1251-labeled untreated 8-galac- tosidase, complete chymotrypsin digestion; R, "'I-PITC/trifluoroa- cetic acid-treated 8-galactosidase, complete chymotrypsin digestion.

End Labeling of Proteins 15575

45 -

2 9 -

2 5 -

1 7 -

8 -

I G l r p

I

1

I

2 5 0

I

5 0 0

I

750

I

1021

FIG. 4. Analysis of peptide mapping by partial proteolysis of end-labeled @-galactosidase. Samples of '"I end-labeled 8-galactosidase were incubated with proteases and chemical cleavage reagents as outlined under "Materials and Methods" and were run on a 12% polyacrylamide Laemmli gel with fragments being visualized by autoradiography. Autoradiography was done without an intensifying screen at room temperature to improve resolution. A, autoradiograph of peptide map: lane 0, no proteolytic treatment; lane T, trypsin, 10 min; lane V, S. aureus protease, 10 min; lane C, CNBr, 3 h; lane D, DTNB/KCN, 16 h; lane H, hydroxylamine, 16 h; lane N , N - chlorosuccinimide, 30 min. B, comparison of predicted and actual amino acid maps of @-galactosidase. By sizing the fragments of a given proteolysis pattern, an amino acid map can be constructed and compared with the map predicted by the amino acid sequence. Residue number was determined by dividing the molecular weight of the fragment by 110, the average molecular weight of an amino acid residue. G, gel-predicted map; A , actual map from sequence; Asn-Gly, hydroxylamine reaction; Trp, N-chlorosuccinimide reaction; Met, CNBr reaction; Cys, DTNB/ KCN reaction.

electrophoresis. The large amount of label in the upper left corner of each peptide map is due to residual amounts of free reagent present.

This result implies that in a complete chymotrypsin diges- tion of radiolabeled protein pretreated with PITC/trifluo- roacetic acid, almost all of the label is associated with one peptide. These data, the quantitation of label incorporation, and the dansylation analysis argue strongly that the amino terminus is the only free amino group after PITC/trifluoroa- cetic acid treatment and upon labeling with an amino group- specific reagent the amino terminus is specifically labeled.

Peptide Mapping by Partial Proteolysis of End-labeled p- Galactosidase-This procedure was done in a way similar to the method of Cleveland et al. (1977). Since only the amino terminus of the protein is labeled, however, only the fragments containing the amino terminus were detected by autoradiog- raphy. Therefore, the peptide maps of the products of partial proteolysis with reagents specific for a given amino acid would reveal the positions of that amino acid in the sequence, within the resolution of SDS-PAGE. The autoradiograph of such an analysis for @-galactosidase is shown in Fig. 4A. With no treatment, end-labeled p-galactosidase appears largely as a discreet band a t M, = 116,000 implying a negligible amount of internal nicking during the trifluoroacetic acid treatment. Upon treatment with the various proteases and chemical reagents, a unique series of bands are produced for each of the different reactions, obtained reproducibly under identical conditions. There is a large difference between patterns ob- tained for S. aureus protease and hydroxylamine cleavage of radiolabeled @-galactosidase as compared to radiolabeled

PITC/trifluoroacetic acid @-galactosidase,2 consistent with the view that PITC/trifluoroacetic acid-treated material pos- sesses label only at the amino terminus.

By measuring the molecular weights of the bands from the autoradiograph of the SDS gel, a map of predicted amino acid positions can be constructed as shown in Fig. 4B for aspara- gine-glycine (hydroxylamine), tryptophan (NCS), methionine (CNBr), and cysteine (DTNB/KCN). The similarity in the predicted positions and the actual positions as determined from the amino acid sequence (Fowler and Zabin, 1977) is very good for hydroxylamine cleavage. The only extraneous band predicted by the gel pattern may be due to an Asp-Gly site in position 860 or to a labile Asp-Pro site in position 800. The agreement for methionine, cysteine, and tryptophan is also quite good, although some differences exist. The presence of many sites would naturally decrease the ability to resolve discreet bands. In general, positions predicted by gel analysis correspond to actual sites in the primary structure with an error of approximately +/-lo% of the molecular weight of the peptide. The correspondence is better for reactions done at basic pH. The procedure may not cleave a t every potential site since even in the denatured state there may exist differ- ences in the reactivity of sites due to nearest neighbor effects or partial folding of the polypeptide. This leads to a problem with vastly differing intensities of bands on the autoradi- ograph. In fact, it should be noted that the linear maps presented are a result of several different exposures. In addi- tion, peptides smaller than 50 residues cannot be resolved

D. G. Jay, unpublished data.

15576 End Labeling of Proteins

B I I I I I A

1 7 2 2 ""'I 11

6.7

2.4

-

I I I I , 1 5 0 100 150 210

FIG. 5. Analysis of peptide mapping by partial proteolysis of end-labeled 434 X immunity repressor. Samples of Iz5I end-labeled X immunity 434 repressor, incubated with proteases and chemical cleaving reagents as outlined under "Materials and Methods," were run on a 16% polyacrylamide Laemmli gel and the fragments were visualized by autoradiography without intensifying screens. A, autoradiograph of peptide map: lane 0, no proteolytic treatment; lane T, trypsin, 30 min; lane C, CNBr, 3 h; lane D, DTNB/KCN, 16 h; lane H , hydroxylamine, 16 h; lane N, N-chlorosuccinimide, 1 h. B, comparison of predicted and actual amino acid maps of X immunity 434 repressor. By sizing the fragments of a given proteolysis pattern, an amino acid map can be constructed and compared with the map predicted by the amino acid sequence. Residue number was determined by dividing the molecular weight of the fragment by 110, the average molecular weight of an amino acid residue. Asn-Cly, hydroxylamine reaction; Met, CNBr reaction; Cys, DTNB/KCN reaction; Trp, N-chlorosuccinimide reaction; Arg, trypsin.

from the dye front while peptides larger than 900 residues cannot be resolved from the undigested P-galactosidase band.

Since there are so many sites for aspartate and glutamate (231) and arginine (66) in the sequence, the usefulness of their positional maps is somewhat limited and they were not included. High frequency amino acids such as these may be useful in demonstrating differences between similar proteins.

Peptide Mapping by Partial Proteolysis of End-labeled X Immunity 434 Repressor-The same procedure was applied to the 434 X immunity repressor, a protein of 210 amino acids whose sequence is known." The data are presented in Fig. 5A with the predicted linear map in Fig. 5B. The advantage of a smaller protein is that fewer specific amino acid sites are present, resulting in simpler patterns for analysis. The sim- plicity of the patterns allows for a more precise localization of the amino acid sites in the sequence. However, the size of the protein is such that resolution of the peptides resulting from proteolysis is not as sharp.

There are 5 Asn-Gly sites in the protein, all of which are predicted by the gel analysis within the error of the experi- ment. Of the 4 methionines present, 3 are accurately localized. In addition, the relative positions of 2 of the 3 cysteines, 4 of the 5 tryptophans, and 4 of the 8 arginines are predicted. There are 2 cysteines in positions 93 and 96 which could not be resolved by SDS-PAGE and may correspond to the band in position 115, although this position is 20% higher than it should be. There also appears to be an anomalous band

R. Yocum, J. Anderson, and M. Ptashne, manuscript in prepa- ration.

corresponding to residue 70. For the tryptophan map, frag- ments a t positions 118 and 141 may both migrate anomalously high and correspond to tryptophan residues 108 and 120, or the observed fragment a t position 118 corresponds well to tryptophan residue number 120 and the fragment a t position 141 is anomalous. It should be noted that two of the arginine residues (6 and 11) would produce peptides too small to be resolved, while a fragment resulting from cleavage at arginine 206 would not be resolved from the intact protein. The argi- nine gel pattern also has an anomalous band a t residue 125. This may correspond to a lysine (residue 122) which may be cleaved by trypsin despite the fact that no unmodified lysine could be detected by dansylation. It should be noted that there is labeled material at the top of the gel and a labeled band at M , = 45,000. These correspond to aggregate and dimer of X immunity 434 repressor, respectively, and do not seem to affect the partial proteolysis pattern significantly.

In general, the patterns show that the procedure offers strong predictive values for the approximate location of spe- cific amino acids in the sequences of proteins, although the possibility of anomalous bands appearing cannot be excluded.

Analysis of X Immunity 434 Repressor: Wild-type and Site- specific Mutant-A site-specific mutant of X immunity 434 repressor has a change in residues 33 to 39 from the sequence Glu-Gln-Leu-Glu-Asn-Gly-Lys for the wild-type protein to the sequence Gln-Leu-Ile-Glu-Ala-Gly-Val for the mutant protein. One of these changes, a glutamate to a glutamine a t position 33, leads to the loss of an S. aureus protease site. Wild-type and mutant X 434 repressor were PITC/trifluo-

End Labeling

roacetic acid-treated, radiolabeled, and subjected to proteol- ysis with trypsin and S. aureus protease. The results of this experiment are shown in Fig. 6.

The S. aureus protease cleavage pattern for wild-type and mutant species are identical except for the absence of a band a t 4000 Da corresponding to a peptide of 36 residues in the mutant lane (Fig. 6, E and F). This value is close to the predicted size of 33 residues. The patterns for mutant and wild-type proteins with trypsin are virtually identical.

These data demonstrate that this positioning procedure can be used to distinguish between very similar proteins differing in one amino acid. It can not only demonstrate the difference but also it can predict the location within the resolution of SDS-PAGE. This procedure could be useful in comparing homologous proteins, locating variable and constant domains in the primary structure. It should be noted that since all cleavage sites may not be detected, a positive result of a change in partial proteolysis pattern would be informative while a negative result may not be.

CONCLUSIONS

A general method for the specific labeling of the amino terminus of proteins and its application in the positioning of certain amino acids in the sequence has been presented. The specific labeling of the amino terminus has been demonstrated by: 1) the absence of dansyl lysine and the presence of the dansylated second amino acid of the sequence in the PITC/ trifluoroacetic acid-treated protein; 2) the quantitation of label incorporated into PITC-treated, PITC/trifluoroacetic acid-treated and untreated protein; 3) the presence of a single radiolabeled spot in two-dimensional peptide maps of radio- labeled PITC/trifluoroacetic acid-treated @-galactosidase compared to the presence of many spots in the radiolabeled untreated material. Furthermore, the patterns of partial pro- teolysis of radiolabeled PITC/trifluoroacetic acid-treated p-

A B C D E F

2 2 .

2.4 .

of Proteins 15577

galactosidase correspond well to the predicted patterns from the amino acid sequence while the results obtained with radiolabeled unprotected material show no relationship to the amino acid sequence. All these points strongly argue for the specific labeling of the amino terminus of &galactosidase. Repeating the procedure for X immunity 434 repressor and transferrin (data not shown) demonstrates that the method can be generally applied to proteins.

It has been demonstrated that peptide mapping of the products of partial proteolysis of an end-labeled protein can be used to predict the positions of specific amino acids along the sequence and that more accurate predictions can be made when the protein possesses relatively few sites, as illustrated by the hydroxylamine digestions, or for smaller proteins as shown by peptide maps of X immunity 434 repressor protein. By comparison of the peptide maps of the products of partial proteolysis of two similar proteins (wild-type and mutant X immunity 434 repressor), a single amino acid difference be- tween the two proteins was identified.

This procedure has applications beyond those demonstrated in this paper. By comparison peptide mapping of an end- labeled protein and the same protein labeled at another site, the position of the other site may be localized by observing the loss of coincidence of bands in the two gel electrophoresis patterns. The largest fragment possessing the end label with- out the second label defines the position of the site of the second label. This analysis may best be applied by the use of differential double labeling, electrophoresis, and counting the gel slices. This procedure could be used for covalent active site inhibitors, sites of phosphorylation, sites of glycosylation, and, by use of the method in tandem with immunoblotting, sites of monoclonal antibody binding. This would provide a rapid and simple way to map the location of important sites in the sequences of proteins.

There are, however, a number of limitations to the proce- dure. 1) The limit of resolution of SDS-PAGE does not allow one to resolve fragments differing in size by only a few residues, thus only approximate positions can be obtained. 2) There are specific cleavages at only some of the amino acids. 3) There must be a free amino terminus on the protein. 4) Not all sites will be recognized. Differences in reactivity of sites due to partial folding or nearest neighbor effects will lead to inability to detect all potential sites. 5) Due to heter- ogeneity of amino acid side chains, anomalous migration of a peptide in SDS-PAGE, although rare, can occur. 6) Lysines are modified and thus cannot be cleaved.

These limitations aside, the procedure coupled with other methods in protein chemistry will be a useful tool in the elucidation of protein structure.

Acknowledgments-I wish to thank Guido Guidotti in whose labo- ratory this work was conducted for his enthusiastic support and advice; Robin Wharton for the generous gift of mutant and wild-type X 434 repressor; and Gilbert Chin and Jonathan Drachman for helpful discussions.

FIG. 6. Comparison of peptide maps of partial proteolysis products of wild-type and mutant 434 repressor. Samples of ' 9 end-labeled wild-type and mutant 434 repressor were treated with trypsin or S. aureus protease for 30 min. Digested samples were run on a 16% Laemmli gel and fragments were visualized by autradiog- raphy. As controls, samples were also given no proteolytic treatment. Lane A , mutant, no proteolytic treatment; lane B, wild-type, no proteolytic treatment; lane C , mutant, trypsin; lane D, wild-type, trypsin; lane E , mutant, S. aureus protease; lane F, wild-type, S. aureus protease.

REFERENCES Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529-539 Bornstein, P. (1970) Biochemistry 12, 2408-2421 Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U.

Dion, A. S., and Pomenti, A. A. (1983) Anal. Biochem. 129,490-496 Drapeau, G. R., Boily, Y., and Houmard, J. (1972) J . Biol. Chem.

Fowler, A. V., and Zabin, I: (1977) Proc. Natl. Acad. Sei. U. S. A. 74,

Gray, W. R. (1972) Methods Enzymol. 25, 121-138 ,

Gross, E. (1967) Methods Enzymol. 11, 238-251

K. (1977) J. Biol. Chem. 252,1102-1106

247,6720-6726

1507-1510

15578 End Labeling of Proteins

Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman, T. C.

Konigsberg, W. (1967) Methods Enzymol. 11,461-469 Laemmli, U. K. (1970) Nature (Lond.) 2 2 7 , 680-685 Landon, M. (1977) Methods Enzymol. 4 7 , 145-149 Markowitz, S., and Marchesi, V. T. (1981) J. Bwl. Chem. 256,6463-

Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 6 5 , 499-

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad.

Schenkein, I., Levy, M., Franklin, E. C., and Fragione, B. (1977)

(1973) J. Biol. Chem. 248,6583-6591

6468

560

Sei. U. S. A. 74,5463-5467

Arch. Biochem. Biophys. 182 , 64-70

Shechter, Y., Patchornik, A., and Burstein, Y. (1976) Biochemistry

Smyth, P. G. (1967) Methods Enzymol. 11,214-231 Tsung, C. M., and Fraenkel-Conrat, H. (1965) Biochemistry 4 , 793-

Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 Weiner, A. M., Platt, T., and Weber, K. (1972) J. Biol. Chem. 2 4 7 ,

Wilchek, M., and Witkop, B. (1967) Biochem. Biophys. Res. Commun.

Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal.

Yoshimoto, T., Walter, R., and Tsuru, D. (1980) J . Biol. Chem. 255 ,

15,5071-5075

801

3242-3251

2 6 , 296-300

Biochem. 118 , 197-203

4786-4792