Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

The denaturation of covalently inhibited swine pepsin.

Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.     

Effect of pepsin pretreatment on pulse-cytophotometric DNA histograms.

Samples of mitotic L-cells were investigated after different preparation and staining procedures using the technique of pulse-cytophotometry. It is shown that most mitotic cells which should appear in the second peak of the DNA histogram are disintegrated or separated into halves by pepsin pretreatment. Hence, the designation 'G2 + M' for the second peak is not correct for this preparative method. This should be taken into account in cell kinetic investigations performed after pepsin pretreatment.     

Synthesis of dideoxy-pepstatin. Mechanism of inhibition of porcine pepsin.

Dideoxypepstatin has been synthesized and shown to be a competitive inhibitor of pepsin (K_i = 2.1 × 10^?7 M).     

Inhibition of pepsin by polyions and c.d. studies

The effects of poly(vinyl sulphate) (PVS), poly(vinyl pyrrolidone) (PVP) and protamine sulphate with the enzyme pepsin (EC 3.4.23.1) have been investigated. PVS, PVP and protamine acted as inhibitors of the enzyme pepsin at low concentrations, but at high concentrations of the polyions (with the exception of PVS) the inhibition was less pronounced. The catalytic effectiveness of several polyions has been shown experimentally; when used at pH 2.1 with haemoglobin and at pH 5.0 with azocasein thus acted as a weak proteolytic catalyst. The order of effectiveness was: polybrene greater than poly(L-lysine) (PLL) greater than spermine greater than spermidine greater than protamine greater than and PVP relative to pepsin. The hydrolysis of the substrates by the enzyme decreases in the presence of high concentrations of monovalent and/or divalent salts. The purity of the enzyme was assessed by determination of the molecular weight using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), phosphorus and nitrogen content. The circular dichroism (c.d.) spectrum of the enzyme in the wavelength range 240-310 nm, where the polycations did not have a c.d. spectrum has also been studied. Each of the polyions had a definite effect on the c.d. spectrum, showing strong binding to the enzyme. The neutral polymer PVP was also found to modify the c.d. spectrum, showing strong binding to the enzyme. The strengths of the interactions as indicated by the magnitudes of the changes in the c.d. spectrum of pepsin at 270 nm were: polybrene greater than protamine greater than PLL greater than PVP greater than spermidine greater than spermine.     

Kinetic studies on the mechanism of pepsin action.

The linear noncompetitive inhibition of the pepsin-catalyzed hydrolysis of Ac-Phe-Phe-Gly at pH 2.1 by L-Ac-Phe, L-Ac-Phe-NH2, and L-Ac-Phe-OEt has been claimed to substantiate the ordered release of products specified by the amino-enzyme mechanism for pepsin action. According to this interpretation, the binding of inhibitor to free enzyme and the amino-enzyme intermediate (Scheme I) generates the observed inhibition pattern. The proposition is valid only if a simple alternative explanation for the kinetic data, Scheme II, can be disproved. Scheme II attributes the inhibition pattern to the binding of inhibitor to free enzyme and the enzyme-substrate (Michaelis) complex. The experiments reported here have enabled us to distinguish between the two mechanisms. The pepsin-catalyzed hydrolyses of Ac-Phe-Trp, Z-H'IS-Phe-Trp, Z-Gly-His-Phe-Trp, and Z-Ala-His-Phe-Trp at pH 1.8 occur exclusively at the Phe-Trp bond and must yield the same amino-enzyme, E-Trp, if it is implicated. Under these circumstances, Scheme I requires that a plot of 1/kc vs. (I)o for the four substrates and a given noncompetitive inhibitor provide a set of four parallel lines. Scheme II predicts that the four lines generally will not be parallel. L-Ac-Phe, L-Ac-Phe-NH2, L-Ac-Phe-OMe, and D-Ac-Phe act as linear noncompetitive inhibitors for the pepsin-catalyzed hydrolysis of the four Trp-containing substrates. The plot of 1/kc vs. (I)o for each inhibitor results in a set of four nonparallel lines. Therefore Scheme II must be correct and the detection of noncompetitive inhibition accompanying the pepsin-catalyzed hydrolysis of peptides offers no insight into the merits of the amino-enzyme hypothesis.