Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Abstract

Reactions of human plasma α 1-antitrypsin ( α 1-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α 1-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (ϵ-NH 2 groups of lysine residues) in human plasma α 1-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α 1-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α 1-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α 1-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α 1AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α 1-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α 1-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α 1-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α 1AT, all of the other chemically modified α 1-AT derivatives studied presently retained their immunological reactivities against antisera to native α 1-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α 1-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α 1-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α 1-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α 1-AT molecule. Based on these modification studies, it is concluded that α 1-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α 1-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).