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Isolation and Identification of Large Overlapping Fragments of Rabbit Myelin Basic Protein Produced by Limited Peptic Hydrolysis
Author(s) -
Martenson Russell E.,
Law Mona J.,
Deibler Gladys E.,
Lüthy Vivian
Publication year - 1981
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1981.tb06319.x
Subject(s) - chemistry , peptide , chromatography , size exclusion chromatography , hydrolysis , cleavage (geology) , urea , pepsin , peptide bond , acetic acid , biochemistry , enzyme , biology , paleontology , fracture (geology)
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 Phe 44 ‐Phe 45 , Phe 87 ‐Phe 88 , Leu 109 ‐Ser 110 , and Leu 151 ‐Phe 152 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 Leu 109 ‐Ser 110 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.