Biochemistry 1986, 25, 5425-5432 5425 Wolodko, W. T., OConnor, M. D., & Bridger, W. A. (1981) Wolodko, W. T., Brownie, E. R., O'Connor, M. D., & Bridger, Weitzman, P. D. J., & Kinghorn, H. A. (1978) FEBS Lett. Wolodko, W. T., Brownie, E. R., & Bridger, W. A. (1980) 88, 255-258. J. Bacteriol. 143, 231-237. Proc. Natl. Acad. Sci. U.S.A. 78, 2140-2144. W. A. (1983) J. Biol. Chem. 258, 14116-14119. New Hydrophilicity Scale Derived from High-Performance Liquid Residues with Antigenicity and X-ray-Derived Accessible Sites? Chromatography Peptide Retention Data: Correlation of Predicted Surface J. M. R. Parker,$ D. Guo, and R. S. Hodges* Medical Research Council Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received September 19, 1985; Revised Manuscript Received April 25, I986 ABSTRACT: A new set of hydrophilicity high-performance liquid chromatography (HPLC) parameters is presented. These parameters were derived from the retention times of 20 model synthetic peptides, Ac- Gly-X-X-(Leu)3-(Lys)2-amide, where X was substituted with the 20 amino acids found in proteins. Since hydrophilicity parameters have been used extensively in algorithms to predict which amino acid residues are antigenic, we have compared the profiles generated by our new set of hydrophilic HPLC parameters on the same scale as nine other sets of parameters. Generally, it was found that the HPLC parameters obtained in this study correlated best with antigenicity. In addition, it was shown that a combination of the three best parameters for predicting antigenicity further improved the predictions. These predicted surface sites or, in other words, the hydrophilic, accessible, or mobile regions were then correlated to the known antigenic sites from immunological studies and accessible sites determined by X-ray crystallographic data for several proteins. It is now thought that the entire surface of a protein can be considered antigenic when peptide fragments of the protein surface are used as immunogens or a different species is used as the immunizing host (Green et al., 1982; Benjamin et al., 1984). Antigenic sites are defined as those residues of a native protein that are bound by antibodies raised to a native protein, native protein fragments, or synthetic peptides. By definition, since antigenic sites are those recognized by antibodies, it is most likely that these sites are accessible or on the surface of a protein, and these regions are probably more mobile than interior regions. Since these sites are on the surface,-they are probably hydrophilic. Indeed, algorithms for hydrophilicity and accessibility have been used to predict antigenicity. We have experimentally determined a new hydrophilicity scale derived from the contribution in high-performance liquid chromatography (HPLC)' of each amino acid side chain to the retention time of model synthetic peptides, Ac-Gly-X- X-(Leu),-(Lys),-amide, where X was substituted by the 20 amino acids found in proteins. This new set of hydrophilicity parameters was used in a modified Hopp and Woods (1978) algorithm to predict which areas of a protein are on the sur- face. It was found that these predicted surface sites correlate well with the known antigenic sites for myoglobin, lysozyme, cytochrome c, and influenza hemagglutinin. An excellent correlation was also shown for accessible sites determined by This work was supported by the Medical Reseavch Council of Can- ada and equipment grants from the Alberta Heritage Foundation for Medical Research. * Correspondence should be addressed to this author. *Recipient of an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship. X-ray data for myoglobin, lysozyme, cytochrome c, bovine trypsin, rat mast cell protease, and Streptomyces griseus trypsin. EXPERIMENTAL PROCEDURES Muterials. Unless otherwise stated, chemicals and solvents were reagent grade. Diisopropylethylamine (DIEA), di- chloromethane (CH,Cl,), and trifluoroacetic acid (TFA) were redistilled prior to use. Picric acid was dissolved in CH2Clz and dried over magnesium sulfate. Acetonitrile (HPLC grade) was obtained from Fisher Scientific, Fairlawn, NJ. Double- distilled water was purified by passage through a Milli-Q water purification system (Millipore Corp., Bedford, MA). Poly- (styrene-co-divinylbenzene) benzydrylamine hydrochloride resin (0.75 mmol of NH2/g of resin) and poly(styrene-co- divinylbenzene) chloromethyl resin ( N 1 .O mmol of Cl/g of resin) were purchased from Beckman Instruments, Inc., Palo Alto, CA, and Pierce Chemical Co., Rockford, IL, respectively. tert-Butyloxycarbonyl (Boc) amino acids were purchased from Vega Biochemicals (Tucson, AZ), Bachem Fine Chemicals, Inc. (Torrance, CA), Beckman Instruments, Inc. (Palo Alto, CA), and the Protein Research Foundation (Japan). Apparatus. Peptide synthesis was carried out on a Beckman peptide synthesizer, Model 990. The HPLC instrumentation was composed of a Spectra-Physics SP8700 solvent delivery system and SP8750 organizer module, combined with a ' Abbreviations: HPLC, high-performance liquid chromatography; RPC, reversed-phase high-performance liquid chromatography; DIEA, diisopropylethylamine; TFA, trifluoroacetic acid; BOC, tert-butyloxy- carbonyl. 0 1986 American Chemical Societv5426 BIOCHEMISTRY PARKER ET AL. 0.08 c R Io 2 OD8 0 Ac-Gly-X-X -(Leu)< (Lys)-amlda I",', ,,''I., ,'I"' ~""~"'"' 15 25 35 45 ELUTION TIME (mln) FIGURE 1: Representative RPC profile at pH 7.0 of synthetic peptides with the sequence Na-acetyl-Gly-X-X-(Leu),-(Lys),-amide, where X is substituted by the 20 amino acids found in proteins. Elution profiles were obtained with a SynChropak RP-P C-18 column (250 X 4.1 mm i.d.) using a linear AB gradient of 1.67% solvent B/rnin and a flow rate of 1 mL/min. Solvent A was aqueous 10 mM (NH4),HP04/0.1 M NaC10, buffer, and solvent B was 0.1 M NaCIO, in aqueous acetonitrile (H20/CH3CN, 40:60). Hewlett-Packard HP 1040A detection system, HP3390A in- tegrator, HP85 computer, HP9121 disk drive, and HP7470A plotter. Samples were injected into a 500-pL injection loop (Model 7125, Rheodyne Inc., Cotati, CA). Columns. Peptide retention times were determined on a reversed-phase SynChropak RP- 18 (C- 18) column (250 X 4.1 mm i.d., 6.5-pm particle size, 300-A pore size, carbon loading -7.5%). Peptide Synthesis. The peptide analogues
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