Isolation and Identification of Large Overlapping Fragments of Rabbit Myelin Basic Protein Produced by Limited Peptic Hydrolysis

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Abstract
Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 m-ammonium formate (pH 6.00) for 15–20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion-exchange chromatography and gel filtration and identified by amino acid and COOH-terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44-Phe45, Phe87-Phe88, Leu109-Ser110, and Leu151-Phe152 bonds were isolated: peptides (1–151), (1–109), (1–87), (45–168), (45–151), (45–109), (88–168), (88–151), and (110–168). Of these, peptides (1–151), (1–87), and (88–151) were recovered in the greatest yield (0.14–0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1–109) and (110–168), indicating that the Leu109-Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe-Phe and Leu-Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1–44), (1–28), (45–87), (88–109), (110–151), and (152–168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above-mentioned peptides, together with the previously isolated peptides (1–14) and (15–44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 m-acetic acid and 8 m-urea.