Modification of Carboxyl Groups in Pepsin

The chemical modification of pepsin carboxyl groups using N ‐(2,4‐dinitrophenyl)‐hexamethylenediamine or compound (I) in the presence of a water‐soluble 1‐cyclohexyl‐3‐[2′‐( N ‐methylmorpholino)‐ethyl]‐carbodiimide p ‐toluenesulphonate or compound II at pH 5.5 resulted in the incorporation of one molecule of I per molecule of pepsin with a concomitant drop of activity to about 40% when measured against hemoglobin and 50% against N ‐acetyl‐ l ‐phenylalanyl‐ l ‐tyrosine as substrates. It was established that the modification of three carboxyl groups took place: β‐carboxyl group of the aspartic acid residue, γ‐carboxyl group of the glutamic acid residue and the α‐carboxyl group of C‐terminal alanine. It is assumed that this reaction is preceded by the hydrophobic binding at some site, specific for the pepsin hydrophobic zone, with a subsequent reaction of I with the carboxyl groups localized near this zone.