Abstract
The spectral properties of Bowman-Birk soybean trypsin inhibitor (BBI) were investigated by analyzing difference absorption spectra and difference CD spectra and by comparing them with those of tyrosyl model compounds. The O-acetylation of tyrosyl side chains showed that the ultraviolet CD bands of BBI above 225 nm originate from disulfide side chains and tyrosyl phenolic groups; phenylalanyl residues do not give rise to detectable CD in BBI in this wavelength region. The results of the tyrosyl ionization experiment were consistent with this interpretation. A broad negative CD band centered around 280 nm in BBI arises mainly from disulfide bonds (epsilonL - epsilonR = -0.83 M-1 cm-1 per disulfide). Each of 2 tyrosyl residues gives rise to negative CD in this region; together they contribute approximately 10% of the total CD intensity at 277 nm (epsilonL - epsilonR = -0.36 M-1 cm-1 per tyrosyl). Disulfide bonds in BBI also have a broad positive CD band centered around 240 nm (epsilonL- epsilonR = 0.9 M-1 per disulfide(. Tyrosyl side chains give rise to a sharp positive peak at 231 nm, overlapping with the positive disulfide CD. Dimerization of monomeric BBI did not alter the CD profile. One of two tyrosyl phenolic groups is relatively exposed and can be 0-acetylated by 100- to 1500-fold molar excess of N-acetylmidazole. The other is inaccessible to the reagent even in the presence of 8 M urea, but can be acetylated in the presence of 6 M guanidine hydrochloride. Fully acetylated BBI has the near-ultraviolet disulfide CD and the far-ultraviolet polypetide CD very similar to those of the native inhibitor, indicating the O-acetylation of two tryosyl side chains did not induce much conformational change in BBI. The near-ultraviolet CD of BBI was altered in the presence of 8 M urea of 6 M guanidine hydrochloride, with a greater change brought about by the latter. Dithiothreitol (20 mM) completely abolished the tyrosyl and disulfide CD in this region.
Highlights
This study shows that ultraviolet CD spectrum of BBI above 225 nm consists of cystinyl and tyrosyl origins
Disulfide bonds in BBI have a broad positive CD band centered around nm
Summary
Absorption spectra were recorded on a Cary Model 15 spectro-V photometer. CD spectra were measured with an instrument described elsewhere [12,13,14]. Absorption spectra were recorded on a Cary Model 15 spectro-. To obtain difference CD spectra, each CD spectrum was stored in a signal averager and recorded separately before subtracting one spectrum from the other. Crude BBI was extracted from defatted.soybean flour (Nutrisoy 7-B). The inhibitor was further purified by gel filtration on a Sephadex G-100 column (3 x 43 cm) [4]. The purified BBI was dialyzed against cold water, lyophilized and stored at 46 until used. The final yield was 367 mg BBI from 1.5 kg defatted soybean flour. Homogeniety of the purified preparation was ascertained with the polyacrylamide gel disc electrophoresis [17]
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