Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin.

Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.

optical densities of about 0.8 were used routinely. Blank spectral curves were obtained after every sample scan for each solvent used. The CD data are reported as molar ellipticities [0] based on the monomer molecular weights of the inhibitors.

AND RELATED DlSCUSSION
Amino Acid Analysis-The amino acid compositions of BEPCI and BEPTI are shown in Table I. They show the characteristic features of the low molecular weight protease inhibitors from legume seeds, including high cystine (greater than 15% by weight), 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 BEPCI uersus 8/16 for BEPTI, and the presence of the single methionine in BEPTI and the second phenylalanine of BEPCI. The minimum molecular weights calculated from the amino acid analysis, 8950 for BEPCI and 8200 for BEPTI, agree with results from sodium dodecyl sulfate gel electrophoresis, which indicated monomer molecular weights under 10,000, with BEPCI slightly larger than B,EPTI (5). BEPCI and BEPTI differ more from each other than from pure isoinhibitors found in the garden bean (8). BEPCI is amazingly similar to garden bean isoinhibitor IIIb in amino acid composition and number; both are chymotrypsin inhibitors with double-headed activity. BEPTI strongly resembles garden bean isoinhibitor I in size and amino acid composition; both inhibit trypsin. but not chymotrypsin. BEPCI and BEPTI both contain 14 half-cystine residues. Treatment with dansyl chloride by the urea method of Gros and Labouesse (9) showed that the inhibitors contain no free sulfhydryl groups. The presence of seven cross-linking disulfides in a chain of about 80 residues demands a compactly folded molecule, involving many chain bends. BEPCI and BEPTI are rich in serine and aspartic acid residues, which have been shown to be important in chain bends (10). Using the classification of polar and nonpolar residues defined by Hatch (ll), BEPCI has a polar/nonpolar ratio of 3.0, and that of BEPTI is 2.47. Such very high ratios are indicative of large surface to volume ratios in proteins.
Near-ultraviolet Absorption Spectra-The ultraviolet absorption spectra of BEPCI and BEPTI are shown in Fig. 1. The absence of a typical protein maximum around 280 nm results from the high disulfide (t,,, 245 nm) and low aromatic content of the inhibitors. The observed molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI computed from the minimum molecular weights described above. They remain unchanged from pH 3 to 8.
Phenylalanine does not contribute significantly to absorbance at 280 nm, and cZsO of the single tyrosine per inhibitor is expected to be only about 1200 (12). Since the inhibitors contain no tryptophan, it appears that there is an anomalously high disulfide contribution to the absorbance at 280 nm. The magnitude per disulfide is about twice the value seen in L-cystine (13). Such long wavelength absorption bands of disulfides may arise from reduced accessibility to solvent (14) or possibly from disulfide dihedral angles less than 90" (15-17). The greater extinction coefficient of BEPTI than BEPCI at 280 nm is not rationalizable in any simple way. Because the inhibitors contain no tryptophan and only a single tyrosine, they allow convenient study of the tyrosyl chromophore. In normal protein spectra the tyrosyl bandwidth is so large that it is difficult to obtain information about individual chromophores. Derivative spectra assist investigation of the tyrosyl chromophore (2). Derivative spectra of BEPCI were indistinguishable in water, 10m3 M HCl, or pH 8.0, 0.1 M Tris-HCl. The derivative spectrum of BEPTI was slightly different from that of BEPCI, but again was the same for all three solvents. Fig. 2 (2), where R is the ratio of the intensity of the major derivative peak to that of the minor peak and AX,+ is the half-width of the major band. Both of these quantities increase when there is spectral heterogeneity due to multiple chromophores. Fig. 4  A n-hour exposure to dithiothreitol at room temperature had no effect on the CD spectrum. Ten additional hours at 37" led to substantial changes in the spectrum.
An additional hour at 60" resulted in more dramatic changes. These results are shown in Fig. 7 Optical Properties of Black-eyed Pea Protease Inhibitors measured immediately at pH 8.0 and after 24 hours of exposure to air at 4", pH 8.0 or pH 12.5 (Fig. 8) in the native inhibitor also proceeds very reluctantly. All of these findings suggest that the structure of these two protease inhibitors is unusual and is far more rigid than that of' a typical globular protein.