The use of high voltage electrophoresis in determining the cleavage characteristics of endopeptidases

A tank-type high voltage paper electrophoresis apparatus was constructed and standardized primarily for the separation of amino acids and peptides of protein hydrolysates. Two tanks were used to eliminate the necessity of changing the coolant and electrolyte systems whenever a change in the electrolyte was desired. A series of experiments showed that electrolytes consisting of (pyridine-acetic acid-water, 12.0:0.3:90.0, v/v) pH 6.48 and (88% formic acid-acetic acid-water, 2.0:8.0:90.0, v/v) pH 1.92. were the most effective for the separations required. High voltage electrophoresis techniques were employed in three research problems. By using lysozyme, a protein with a known sequence (partial sequence shown below), the steric effect of large natural residues, -Ser/60-Arg-Trp-Trp-Cys-Asp/65-Asn-Gly- TRP ⁶² and TRP ⁶³ , upon the formation of S-aminoethylated and S-carboxymethylated cysteinyl derivatives at Cys ⁶⁴ ' was determined. In addition, the steric effect of the two tryptophan residues on tryptic hydrolysis as Arg ⁶¹ and AE-Cys ⁶⁴ was determined...