ROLE OF DISULFIDE LINKAGES IN STRUCTURE AND ACTIVITY OF PROTEINASE-INHIBITOR FROM HORSEGRAM (DOLICHOS-BIFLORUS)

Proteinase inhibitor isolated from horsegram (Dolichos biflorus or Mac rotyloma uniflorum) inhibited specifically the enzymes trypsin and chy motrypsin. The inhibitor contained seven disulfide linkages and was fr ee from thiol groups. The inhibitor is resistant to denaturation by ur ea, guanidine hydrochloride or sodium dodecyl sulfate. Reduction of th e inhibitor with dithiothreitol abolished both trypsin and chymotrypsi n inhibitory activities. The kinetic plots of the reduction as followe d by activity and loss in structure as reflected in the 257 nm CD band could be superposed; loss in the activity paralleled the loss in stru cture. The kinetics of the reduction process was complex; reduction of the inhibitor was slow and depended on the concentration of DTT. Redu ction of the disulfide linkages with DTT affected the tertiary structu re significantly and secondary structure was not affected considerably . Fluorescence quenching by acrylamide and potassium iodide suggested the unfolding of the molecule due to reduction. Thus, disulfide linkag es play a predominant role in maintaining the three-dimensional struct ure of the inhibitor.