Stabilization of bovine trypsin by reductive methylation.

Reductive methylation has little or no detectable effect on the catalytic or physicochemical properties of bovine trypsin but reduces its susceptibility to autolysis. Increased stability after methylation appears to result from the conversion of trypsin-susceptible lysine residues to trypsin-resistant epsilon-N,N-dimethyllysine residues. Reductively methylated trypsin is easily prepared and may be useful in place of trypsin where autolysis is otherwise difficult to control.

Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme.

In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.

The reactivity of p-nitrophenyl acetate with serum albumins.

1. Serum albumins from nine of 10 vertebrate species were found to react rapidly with p-nitrophenylacetate. 2. The high reactivities were shown to be partially attributable to strong, rapidly reversible binding of p-nitrophenylacetate by each serum albumin. 3. As previously observed in the case of human serum albumin (Koh and Means, Arch. Biochem. Biophys. 192, 73-79, 1979), this binding takes place in the primary binding site for several physiologically (i.e. tryptophan, small fatty acid anions) and pharmacologically (i.e. diazepam) important compounds. 4. Horse serum albumin differed from all other serum albumins included in this study in that it did not react rapidly with p-nitrophenylacetate, presumably, due to significant differences in its corresponding binding site.

Immobilization of proteins on organic polymer beads.

A new method is described for the immobilization of biologically active proteins onto several types of organic polymer beads. First, the soluble protein is modified by reaction with an excess of a hydrophobic imidoester, for example methyl 4-phenyl-butyrimidate, at ca. pH 9 and 0 degrees . Excess imidoester and side products resulting from imidoester hydrolysis are separated from the hydrophobic protein derivative by exclusion chromatography or dialysis. A suspension of polymer beads (e.g. Amberlite XAD-7) is then added to a solution of the modified protein at room temperature or below and stirred gently for 1-2 h. The polymer beads are allowed to settle, separated from the solution by decantation or filtration, washed, and resuspended in an appropriate buffer. Quantitative adsorption of protein to the polymer beads is observed under such conditions. The synthesis of seven hydrophobic imidoesters and their use for the immobilization of trypsin onto several types of porous polymer beads is described. The immobilizations of trypsin, yeast alcohol dehydrogenase, and E. coli asparaginase by this procedure with high recoveries of catalytic activity, suggests that it will be applicable to a large number of biologically active proteins.

A procedure for the determination of monothiols in the presence of dithiothreitol--an improved assay for the reduction of disulfides.

4,4'-Dipyridyl disulfide (4-PDS) and sodium arsenite have been used to develop a rapid and sensitive assay for monothiols in the presence of dithiothreitol (DTT). The procedure is similar to that of Zahler and Cleland (J. Biol. Chem. 243, 716-719, 1968) but involves the use of a lower pH and 4-PDS, which is more effective than 5,5'-dithiobis-(2-nitrobenzoic acid) at low pH. Background reactions that interfere with the determination of slow reacting thiol groups are much reduced in the new assay procedure. The reduction of oxidized glutathione by DTT at three different pH values and a partial and the complete reduction of bovine serum albumin by DTT have been characterized in order to demonstrate the use of this procedure. In the first two cases monothiol groups were readily detected without interference from DTT. Lower than expected numbers of thiol groups in the case of three reduced and denatured model proteins appeared to reflect their partial complexation by arsenite. Equilibrium and kinetic constants for the formation and dissociation of the DTT-arsenite complex from pH 5 to 8 are presented.

Inactivation of angiotensin converting enzyme by sodium nitroprusside.

Angiotensin I converting enzyme is rapidly inactivated by sodium nitroprusside and that inactivation is suppressed in the presence of chloride ion and by the presence of L-alanyl-L-proline or glycyl-L-tryptophan, which are both competitive inhibitors of its catalytic activity. The inactivation by sodium nitroprusside appears to result from the modification of an unusually reactive lysine residue in or near the active site.

S-nitrosation of serum albumin: spectrophotometric determination of its nitrosation by simple S-nitrosothiols.

The transfer of nitroso groups from S-nitroso-L-cysteine (1) and six other simple S-nitrosothiols to Cys 34 of bovine serum albumin (2) has been followed using Ellman's reagent, 5,5'-dithio-bis (2-nitrobenzoate) (3), to detect the resulting thiols. The described method utilizes the low reactivity of (3) with (2) and the high extinction coefficient of 2-nitro-5-thiobenzoate produced upon its reaction with thiols to follow the transfer of nitroso moieties at low concentrations where other procedures are not feasible. A second-order rate constant of 6400 M-1 s-1 obtained for the reaction of (2) with S-nitrosomercaptoethylamine is approximately 10 times faster than that for its reaction with (1), approximately 40 times faster than that for its reaction with S-nitrosoglutathione, and consistent with Cys 34 being located in a narrow crevice in close proximity to an anionic charge.

Acetylation of human serum albumin by p-nitrophenyl acetate.

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.

5-Nitrosothio-2-nitrobenzoate: a reagent for the nitrosation of thiol groups in proteins.

The S-nitroso derivative of 5-thio-2-nitrobenzoate was synthesized from 5,5'-dithiobis(2-nitrobenzoic acid) and partially characterized. Although relatively unstable, it is easy to prepare and reacts very rapidly with thiols and thiol groups of proteins to give corresponding S-nitrosothiols and 5-thio-2-nitrobenzoate dianion. The latter's easy spectrophotometric detection makes such reactions easy to follow and to quantitate.

Transnitrosation as a predominant mechanism in the hypotensive effect of S-nitrosoglutathione.

S-Nitrosoglutathione, a possible intermediate in the pharmacological mechanisms of many vasodilators, undergoes hemolysis at pH 7.4 and 37 degrees C to give oxidized glutathione and nitric oxide with a second-order rate constant of approximately 3 x 10(-4) M-1sec-1. At the dosages normally employed, this reaction is, therefore, too slow to be an obligatory step in the pharmacological mechanisms of those, usually, fast-acting drugs. Transfers of the nitroso moiety to another thiol or to certain hemoproteins are, however, both much faster and could be involved in those pharmacological mechanisms. Intravenously administered S-nitrosoglutathione reduced the blood pressure of anesthesized dogs and monkeys to the same extent and with essentially the same rapid onset and dissipation as sodium nitroprusside, which is the fastest-acting of those vasodilators.

A spin label study of immobilized enzyme spectral subpopulations.

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site label revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site label of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.