Yoshio Tomimatsu

Physical characterization of ovoinhibitor, a trypsin and chymotrypsin inhibitor from chicken egg white

Abstract A simple method for preparing ovoinhibitor of high specific activity from commercially available chicken ovomucoid is presented. Ultracentrifuge and light-scattering measurements on ovoinhibitor give a weight average molecular weight of 46,500. This, with inhibition data for trypsin and chymotrypsin, indicates an inhibitor to enzyme combining molar ratio of 1:2. Amino acid and chemical analyses of chicken ovoinhibitor show some differences from chicken and turkey ovomucoids, but these do not clearly account for the enzymic differences. Enzymic studies confirm that ovoinhibitor, like turkey ovomucoid but unlike chicken ovomucoid, inhibits trypsin and chymotrypsin, the inhibition of one enzyme being essentially unaffected by the presence of the other.

Cross-linking of α-chymotrypsin and other proteins by reaction with glutaraldehyde☆

Abstract The rate of inactivation of α-chymotrypsin by reaction with glutaraldehyde increased with increasing pH, but the formation of insoluble and active α-chymotrypsin was most rapid at pH 6.2. The pH values for the most rapid glutaraldehyde insolubilization (pH optima) of other proteins were chymotrypsinogen-A, pH 8.2; bovine serum albumin, pH 4.8; soybean trypsin inhibitor, pH 4.8; lysozyme, pH 10.5; and papain, pH 8.6. With the exceptions of α-chymotrypsin and chymotrypsinogen-A, the pH for the most rapid glutaraldehyde insolubilization of these proteins was the same as their isoelectric points. The proteins, which varied in their initial total lysine content, showed the same percentage decrease in lysine upon insolubilization. Insolubilization of α-chymotrypsin was more rapid the lower the ionic strength of the reaction mixture. When the insolubilization was conducted in the presence of the inhibitor, β-phenylpropionate, or with the monoacetyl derivative of α-chymotrypsin, the insolubilized product was more enzymatically active. Concurrent glutaraldehyde insolubilization of bovine serum albumin and α-chymotrypsin was possible only at a pH between the optimum for each protein. An insolubilized cross-linked mercuripapain-α-chymotrypsin was prepared which possessed the activities of each enzyme.

Chicken chymotrypsin and turkey trypsin: Part II: Physical and enzymic properties

Abstract Turkey trypsin, molecular weight 22,500 by light scattering and 18,557 by amino acid analysis, reacted with substrates and inhibitors in the same manner as mammalian trypsins. The enzyme was stabilized by calcium at low and neutral pH. The amino acid analysis also indicated a similarity to mammalian trypsins. Chicken chymotrypsin, molecular weight 20,000 by light scattering and 26,309 by amino acid analysis, exhibited esterase activity almost twice that of bovine alpha chymotrypsin but only two thirds of the protease activity. Calcium did not increase the esterase activity but did stabilize the enzyme near pH 7. At low pH the enzyme was more stable in the absence of calcium. Amino acid analysis indicated chicken chymotrypsin is more like bovine chymotrypsinogen A than bovine chymotrypsinogen B or porcine chymotrypsinogen. The results indicate that natural variations between chicken and mammalian chymotrypsins occur in both physical and enzymic properties, whereas variations between turkey and mammalian trypsins were confined almost entirely to physical properties.

Physical chemical observations on the α-chymotrypsin glutaraldehyde system during formation of an insoluble derivative

Abstract Physical chemical observations have been made on the α-chymotrypsin-glutaraldehyde system during formation of an insoluble, enzymically active product. Lightscattering measurements indicate a two-step reaction, the second step being a linear condensation polymerization reaction. The effects of pH and ionic strength on the rate of the second-step reaction are best explained in terms of an acid shift in the p K a of the ϵ-amino groups of glutaraldehyde-modified lysine residues and a decrease in attractive forces between enzyme particles with increasing ionic strength, respectively. The large cross-linked particles formed appear to be branched flexible coils. Time-dependent ultraviolet spectra demonstrate an apparent hyperchromism due to increased scattering with no new absorption bands. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements show no gross unfolding of the α-chymotrypsin molecule upon cross-linking, although small local changes in conformation could be indicated by changes in the CD spectrum at 255 and 229 nm. Measurements on small soluble polymers show that there is a large loss in activity (60%–70%) during formation of these relatively small derivatives, suggesting that reaction of glutaraldehyde with primary amino groups and not intermolecular cross-link formation could be the main reason for the loss in activity.

Circular dichroism studies of human serum transferrin and chicken ovotransferrin and their copper complexes

Abstract 1. 1. Circular dichroism (CD) measurement of human serum transferrin and chicken ovotransferrin and their copper complexes have been made over the wavelegth range 200–1000 nm. 2. 2. In the protein absorption region the CD spectra of the apoproteins were qualitatively very similar but showed small differences in the magnitudes of their dichroic effects. Copper binding did not cause any significant change in the intrinsic region CD spectra of either transferrin but did affect the aromatic region CD spectra of both transferrins. 3. 3. The absorption bands induced in the near ultraviolet, visible, and near-infrared regions by copper binding were found to be optically active. Differences observed between the CD spectra in this region of the two copper transferring suggest that some dissimilarities exist between the metal-binding sites of the two transferrings. 4. 4. Reults of copper titration studies by CD confirm the presence of two specific, or tight, metal-binding sites. When more than two equivalents of copper are added, evidence is obtained which suggests the presence of additional non-specific, or weaker, binding sites; copper bound to these sites may also induce optically active absorption bands.

Comparison of reflection effects in the brice-type and sofica light scattering photometers: Computer analysis of angular scattering data

Abstract Values of the light scattering parameters for a polystyrene fraction and a dextran sample obtained with the Brice-Phoenix instrument are compared with values obtained with the Sofica photometer. The latter instrument is essentially free from reflection effects. Therefore, the agreement obtained in the values of these parameters by the two instruments provided experimental verification of the procedure used to correct for reflection effects in the Brice-Phoenix instrument. Least squares computer analysis of the angular scattering data served to eliminate the bias inherent in handdrawn Zimm plots.

Resonance Raman spectroscopy of iron (III) - ovotransferrin and iron (III) - human serum transferrin

Abstract We report the resonance Raman spectra in the frequency range 300–1800 cm−1 of Fe (III)-ovotransferrin and Fe (III)-human serum transferrin in aqueous solution at about 10−4M protein concentration. This is the first observation of resonance Raman scattering ascribable to amino acid ligand vibrational modes of a nonheme iron protein. The resonance Raman spectra of the transferrins are similar except that the resonance band near 1270 cm−1 is shifted to a higher frequency for Fe(III)-human serum transferrin than that for Fe(III)-ovotransferrin. The resonance Raman bands observed near 1170, 1270, 1500 and 1600 cm−1 may reflect resonance enhancement of p-hydroxy-phenyl frequencies of tyrosine residues and/or imidazolium frequencies of histidine residues.

Spectroscopic evidence for perturbation of tryptophan in Al(III) and Ga(III) binding to ovotransferrin and human serum transferrin

Ultraviolet spectroscopy has been widely used to study metal binding to transferrins [l-8] . The observed difference spectra are usually interpreted in terms of changes in ionization of tyrosyl residue, although earlier work suggested perturbation [5] or direct involvement [3,9] of tryptophan in Fe(II1) and Cu(I1) binding to transferrins. But Feand Cutransferrins have broad charge-transfer absorption bands in the near ultraviolet [5,10,1 l] which extend into the wavelength region in which the tyrosine and tryptophan chromophores absorb. This makes interpretation difficult. Difference spectra obtained at pH 11 .O have been used to correct for this background absorption of Fe-transferrins [2,4]. But, ionization of tyrosines may not be complete at pH 1 I .O [l] and time-dependent exposure of ‘buried chromophores at alkaline pH [ 121 and alkaline hydrolysis of disulfide bonds [ 131 may further complicate determination of background absorption by this method. Colorless transferrin metal complexes appear to be more suitable for difference spectral studies. Zn(I1) [4,7] and trivalent lanthanides [6] give difference spectra which are similar to that for tyrosine ionization, with no apparent interfering absorption due to the metal ion. Ga(II1) binding to ovotransferrin has been reported [8,14] to give a difference spectrum characteristic of tyrosine ionization. A difference was noted between the spectrum of the first Ga bound and that of the second’ [8] . We have measured, (i) the difference spectra produced by Zn, Ga and Al(II1) binding to ovotransferrin and human serum

Macromolecular properties and subunit interactions of ribulose-1,5-bisphosphate carboxylase from alfalfa

Abstract Fraction 1 protein was extracted from alfalfa ( Medicago sativa ) and the purified protein identified with the enzyme ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). Results of enzyme activity, light-scattering, circular dichroism and differential scanning calorimetric measurements show: 1. 1. The enzyme is quite stable, showing no loss in activity after a years storage, either at 4°C in buffer (0.02 M potassium phosphate, pH 7.5) solution or at −20°C as a precipitate in 60% (NH 4 ) 2 SO 4 . Lyophilization of a salt-free solution of the enzyme produced mostly an insoluble product; lyophilization of a buffer solution of the enzyme produced a soluble product which retained most of its activity. 2. 2. The enzyme molecular weight (light scattering) is 497 000, and in buffer it neither dissociates into subunits at low concentration (6 μg/ml) nor forms molecular aggregates at high concentration (15 mg/ml). Treatment with p- chloromercuribenzoate at pH 7.5 and subsequent storage at pH 9 does not dissociate the subunit structure. 3. 3. In solution at pH 7.5, the enzyme has a compact conformation and helices make up 29% of the secondary structure. At pH 3.4, it is largely unfolded, but the subunits are still associated, and β- and disordered structures dominate the intrinsic region CD spectrum. 4. 4. The enzyme denatures at 76.2°C and has an enthalpy of denaturation of 6.3 cal/g. The presence of a single endotherm suggests that the subunits are tightly bound to each other and are denatured as a single unit.

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Abstract Glutaraldehyde insolubilized chymotrypsin has been prepared by trypsin activation of glutaraldehyde insolubilized chymotrypsinogen-A and found to have esterase activities of 28 and 22% (TEE, ATEE) and proteinase activities of 4.4 and 3.8% (hemoglobin, casein) relative to α-chymotrypsin. A soluble glutaraldehyde crosslinked chymotrypsin, similarly prepared, had a weight average molecular weight of 130,000 and had esterase activity of 90% (ATEE) and proteinase activity of 42% (casein) relative to α-chymotrypsin. These activities were appreciably greater than those shown by glutaraldehyde crosslinked α-chymotrypsin prepared by direct reaction of glutaraldehyde and α-chymotrypsin. Amino acid analyses on the crosslinked enzymes prepared by activation of insoluble and soluble crosslinked zymogens suggest that the insoluble crosslinked enzyme is entirely in the π form and the soluble crosslinked enzyme partly in the π form. Full esterase activity is retained by the trypsin-activated glutaraldehyde insolubilized chymotrypsinogen-A at 25 °C for 14 days. The circular dichroism (CD) changes associated with activation in the native system were also found to occur in the soluble crosslinked system, suggesting that certain conformational changes are necessary for development of enzymatic activity. A CD change at 255 nm toward less negative values was observed when glutaraldehyde was reacted with either native chymotrypsin or crosslinked chymotrypsin generated by activation of glutaraldehyde crosslinked zymogen but not when glutaraldehyde reacted with chymotrypsinogen-A.