Abstract
Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 83 amino acid residues per monomer. Black-eyed pea trypsin inhibitor (BEPTI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 75 residues per monomer. The molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI. The single tyrosyl residue is very inaccessible to solvent in native BEPCI and BEPTI at neutral pH and titrates anomalously with an apparent pK = 12. Ionization of tyrosine is complete in 13 hours above pH 12. No heterogeneity of the local environment of the tyrosyl residues in different subunits can be detected spectrophotometrically. The large number of cystine residues leads to an intense and complex near-ultraviolet CD spectrum with cystine contributions in the regions of 248 and 280 nm and tyrosine contributions at 233 and 280 nm. An intact disulfide structure is required for appearance of the tyrosyl CD bands. The inhibitors are unusually resistant to denaturation when compared with similar low molecular weight proteins of high disulfide content. All observations are consistent with a far more rigid structure for BEPCI and BEPTI than for a typical protein.
Highlights
University, New York, Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds
They show the characteristic features of the low molecular weight protease inhibitors from legume seeds, including high cystine, serine, and aspartic plus glutamic acid content, and little or no tryptophan, tyrosine, phenylalanine, and methionine
The principal differences in the amino acid compositions of the black-eyed pea isoinhibitors can be seen in the ratio of basic to potentially acidic residues, 7/!9 for Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) uersus 8/16 for Black-eyed pea trypsin inhibitor (BEPTI), and the presence of the single methionine in BEPTI and the second phenylalanine of BEPCI
Summary
University, New York, Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan,. The low molecular weight protease inhibitors from many different genera of legumes are quite similar [1] They all contain about 80 amino acid residues with a characteristic composition of little or no tryptophan and methionine, low tyrosine and phenylalanine, and high disulfide content. They are interesting for absorbance and CD studies in the near-ultraviolet region [2,3,4]. ’ The abbreviations used are: BEPCI, black-eyed pea chymotrypsin. and trypsin inhibitor; BEPTI, black-eyed pea trypsin inhibitor; LBI, lima bean trypsin inhibitor; PTI, pancreatic trypsin inhibitor; R x X,, measure of the sharpness of a spectral band; [B], molar ellipticity (degrees cm*/dmol); dansyl, 5.dimethylaminonaphthalene-I.sulfonyl
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