Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives

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Abstract
1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The α-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of β-trypsin retained enzymic activity at values comparable with those of native enzyme tested with α-N-benzoyl-L-arginine ethyl ester and α-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of α-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated β-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus β-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated α-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the α-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of β-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.