Location of SH groups along the polypeptide chain of soybean beta-amylase

The five SH groups of soybean beta-amylase differ in reactivity toward SH reagents such as 2,2'-dithiopyridine (2-PDS), monoiodoacetate and N-ethylmaleimide (NEM). They were designated as SH1, SH2, SH3, SH4, and SH5, in order of their reactivity except for the two buried SH groups, SH4 and SH5. The location of the five SH groups along the polypeptide chain was determined by specific cleavage at the amino side of their cyanocysteine residues which were formed by converting SH to SCN groups by cyanide after modifying the SH groups with 2-PDS. The selective modification of SH groups was achieved as follows: SH1 reacted with 2-PDS at low and high ionic strength, while SH2 reacted only at high ionic strength. SH2 and SH3 were also modified with 2-PDS using SH1-carboxymethylated soybean beta-amylase. The buried SH groups, SH4 and SH5, were modified with 2-PDS under the denaturation conditions after the reactive SH groups, SH1, SH2, SH3, were irreversibly blocked with NEM. On the other hand, the five SH groups were cyanylated with [14C]cyanide or with 2-nitro-5-thiocyanobenzoic acid (NTCB) for the cleavage at all five SH groups. The molecular weight estimation of derivatives of cleaved soybean beta-amylase by SDS-gel electrophoresis showed that the five pairs of fragments (Mw 50,000 & 6,500, 47,000 & 8,000, 38,000 & 18,000, 35,000 & 23,000, and 31,000 & 25,000) were identified with the fragments formed by cleavage at SH1, SH2, SH3, SH4, and SH5, respectively. By considering fragments incorporating 14C (Mw 47,000, 35,000, 25,000, 18,000, and 6,500), the fragments were aligned along the polypeptide chain of soybean beta-amylase, in order from the N-terminus as SH2, SH5, SH3, SH4, and SH1. This order is supported by estimating the molecular weight of fragments formed by high-yield cleavage using NTCB and by analyzing the COOH-terminal residues of the fragment cleaved at SH2.