Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities.
The single disulfide bond in potato Inhibitor I protomers (Mr = 8,000) was reduced and alkylated in the absence of denaturants to the carboxamidomethyl cysteine derivative. Two half-cystines per mol of inhibitor protomer were modified as determined by (a) loss of two sulfhydryl groups per reduced protomer, (b) incorporation of 2 mol of [14C]iodoacetamide per protomer, and (c) amino acid analyses. The alkylated Inhibitor I retained its oligomeric structure (Mr = 40,000) and fully retained its immunological cross reactivity with anti-Inhibitor I serum. Furthermore, the inhibitory properties of modified and unmodified Inhibitor I toward chymotrypsin were identical. The stoichiometric complex between modified Inhibitor I and chymotrypsin was stable at pH 8.0 for over 72 h. The modification of the disulfide bond introduced a lability to both heat and proteolytic enzymes. Ultraviolet difference spectra and near ultraviolet circular dichroism spectra at pH 8.0, between modified and unmodified Inhibitor I, revealed only minor absorption changes due to modification. However, in circular dichroism spectra at pH 2.0, the modified inhibitor exhibited a significant loss of absorption in the region between 270 and 280 nm that was present in the unmodified inhibitor. The cumulative results of this study indicate that the single disulfide bond in Inhibitor I, which forms a rather large disulfide loop between residues 5 and 51, is not important at neutral pH for maintaining major structural conformations which affect immunological or inhibitory activities, but the disulfide bond does impose restraints on the protein which stabilize it toward thermal denaturation and proteolysis. The susceptibility of the reduced inhibitor to plant sulfhydryl enzymes may have some importance in in vivo degradation.
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D003429:Cross Reactions; D004220:Disulfides / Q000032:analysis; D007106:Immune Sera; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000494:pharmacology*; D010944:Plants / Q000032:analysis*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D004220:Disulfides; D007106:Immune Sera; D046911:Macromolecular Substances; D010940:Plant Proteins; C023578:proteinase inhibitor I (plants); D002918:Chymotrypsin