Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin.

Reactions of the sulfhydryl groups of bovine rhodopsin in rod outer segment membranes have been investigated using 4,4'-dithiopyridine. This reagent is uncharged at neutral pH and rapidly equilibrates across phospholipid bilayers. Membrane-bound rhodopsin has two kinetically distinguishable sulfhydryl groups reactive to the reagent, this stoichiometry being unchanged by bleaching provided the sulfhydryl reactions themselves are carried out in the dark. The rates of the reactions, however, are substantially increased by bleaching. Irradiation of bleached membranes, either with white light or wavelengths in the neighborhood of 475 nm, results in an increase in the number of reactive sulfhydryls relative to that found for bleached membranes in the dark. A component of the light-driven reaction is dependent on the Ca2+ content of the medium.     

Reactions of the amino groups in ribonuclease A: II. Identification of reactive amino groups in ribonuclease

Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pK_ma. It was shown that the lys-41 amino group had a pK_a of 9.03 in TEA buffer. The pK_a values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Reactions of the amino groups in ribonuclease A: I. Kinetic studies

A kinetic analysis has been made of the reaction of the amino groups of ribonuclease A with trinitrobenzene-sulfonic acid. The number of reactive groups and the number of subsets were markedly dependent on the nature and concentrations of the buffer and the pH. Apparent values of pK_a, calculated from the variation of the velocity constants with pH, could, in general, be obtained only for pH values above 7.4. Below this pH the velocity constants were greater than the values calculated from the intrinsic constants. The values of pK_a were in the range of 7.9 – 9.0, which are somewhat smaller than those derived from titration data.The change of behavior of the amino groups with pH is confirmed by a study of the effects of ionic strength on the reactions.The velocity constants generally appear to decrease with increasing concentration of protein.It is shown that there is a close correlation between the pH region in which large changes occur of the reactivities of the amino groups of RNase and the kinetics of the enzyme reaction.     

Reactions of ketones with aromatics in acid media. The effect of trifluoromethyl groups and the acidity media. A theoretical study

The reactions of acetone, 2,2,2-trifluoroacetone and hexafluoroacetone in methanesulfonic (MSA) and triflic acids (TFSA) with benzene have been studied at M06-2X/6-311+G(d,p) level using cluster-continuum model, where the carbonyl group is explicitly solvated by acid molecules. The introduction of a trifluoromethyl group into the ketone structure reduces the activation energy of the tetrahedral intermediates formation due to an increase of the electrophilicity of the carbonyl group and raises the activation and the reaction energies of the C-O bond cleavage in formed carbinol due to the destabilization of the corresponding carbocation. The introduction of the second trifluoromethyl group inhibits the hydroxyalkylation reaction due to a very strong increase of the reaction and activation energies of the C-O bond cleavage which becomes the rate determining step. The most important catalytic effect of TFSA compared to MSA is not the protonation of the ketone carbonyl, but the reduction of the activation and reaction energies of the carbinol C-O bond cleavage due to better protosolvation properties. Even for TFSA no complete proton transfer to carbonyl oxygen has been observed for free ketones. Therefore, the protonation energies of free ketones cannot be considered as a measure of ketone reactivity in the hydroxyalkylation reaction.

Reactions of the amino groups of RNAse A: IV. The effects of protein concentration

A study has been made of the effect of ribonuclease (RNAse) concentration on the properties of the amino groups. The biphasic dependence of pK on pH which has been established (Goldfarb and Martin, Bioorg. Chem.5, 137 (1976)). for 5 μM solution of RNAse also have been shown to occur for 50 μM solutions. In the lower pH range (7.5–8.5) the values of pK obtained with 50 μM solutions were similar to those obtained with 5 μM solutions (pK = 7.5) but the intrinsic constants were smaller. In the higher pH range (8.5–10) the pKs in the more concentrated solutions were larger than those found at the smaller concentration and the intrinsic constants were generally smaller. A quantitative study of the concentration vs k_i relation at pH 7.5 indicated a sigmoid relationship for all of the subsets with a constant maximum value equal to, and less than that at 5 μM RNAse and a constant minimum value above that at 20 μM. Parallel studies with oxidized RNAse gave parallel, although not identical, results from which it is proposed that the concentration effect does not arise totally from the three-dimensional structure of native RNAse.     

Reactions of nitrosobenzene with reduced glutathione.

Nitrosobenzene (NOB) formed acid labile conjugates with reduced glutathione (GSH) and hemoglobin within red cells. In vitro, NOB rapidly reacted with GSH with formation of phenylhydroxylamine (PH), oxidized glutathione (GSSG), and a water-soluble compound identified as glutathionesulfinanilide (GSO-AN). Free aniline (AN), aminophenols and azoxybenzene were not detected. The proportion of PH formed increased with increasing GSH concentration and at higher pH values. Spectroscopic analysis revealed the formation of a labile adduct following a second order reaction (K = 5 x 10(3) M-1 . sec-1 at pH 7.4 and 37 degrees). This reaction was reversible because nearly all NOB could be extracted with ether from the labile intermediate. On the other hand, the labile intermediate was transformed into GSO-AN (with increasing rate at lower pH values) or it was cleaved by GSH with formation of GSSG and PH. Intermediate formation of NOB and thiol radicals was ruled out by analysis of the equilibrium data. A tentative scheme is presented for the proposed reaction mechanism.     

Reactions of lipid-derived malondialdehyde with collagen.

Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.     

Reactions of thrombin-serpin complexes with thrombospondin.

Activated platelets release proteins that form stable complexes with thrombin (J. J. Miller, P. C. Browne, and T. C. Detwiler, Biochem. Biophys. Res. Commun. 151, 9-15, 1988). A working model for the reaction (P. C. Browne, J. J. Miller, and T. C. Detwiler, Arch. Biochem. Biophys. 265, 534-538, 1988) includes a dissociable complex of thrombin with released platelet protease nexin, leading to formation of a nondissociable thrombin-nexin complex that then becomes disulfide linked to thrombospondin. This disulfide-linked complex is converted back to the thrombin-nexin complex by reduction of disulfide bonds. Results that allow elaboration on this model are presented. After longer periods of incubation or after incubation with higher concentrations of thrombin, the amount of thrombin complexed with thrombospondin exceeded the amount of thrombin-nexin complex recovered after reduction of disulfide bonds. When the reaction mixture included inhibitors of formation of the thrombin-nexin complex, a slow formation of the thrombin-thrombospondin complex was observed. It was concluded that there is a nexin-independent as well as the faster nexin-dependent disulfide linkage of thrombin to thrombospondin. Addition of thrombin-antithrombin III complexes to the supernatant solution of activated platelets also led to complexes with thrombospondin, demonstrating that serpins other than platelet protease nexin facilitate incorporation of thrombin into complexes with thrombospondin. By heparin affinity chromatography, it was shown that thrombin-nexin complexes dissociably associate with thrombospondin prior to formation of disulfide-linked complexes. These observations are incorporated into a more detailed model of the reaction.     

Reactions of proteins with ethyl vinyl sulfone.

Ethly vinyl sulfone (EVS) alkylates xi-amino groups of lysine side chains and imidazole groups of histidine residues in proteins. Amino acid analysis of hydrolyzates of EVS-treated polylysine shows that lysine forms two derivatives, presumably xi-N-(ethylsulfonylethyl)lysine and xi, xi, N,N-bis(ethylsulfonylethyl)lysine that are eluted as well-resolved peaks on the (long basic) physiological column of our amino acid analyzer at about 118 and 60 min, respectively. Peaks with identical elution times were also observed after EVS-treatment of BSA and wool. The postulated histidine derivative, presumably N3-im-(ethylsulfonylethyl)histidine is also eluted as a well-resolved peak on the same column at about 90 min. A peak with an identical elution time was observed in a hydrolyzate of EVS-treated polyhistidine. The described alkylation has potential utility for modifying proteins.     

Reactions of the amino groups of RNAse A: III. The existence of pH-dependent values of pK

The rates of the trinitrophenylation of the amino groups of ribonuclease A (RNAse) with the specific reagent trinitrobenzene sulfonic acid have been studied at 27°C, between pH 7.0 and 9.9. From the variation of the velocity constants with pH it has been shown that the reaction is biphasic in the sense that for each amino group two pKs have been found: one (pK = 7.3–7.52) in the range of pH between 7.0 and 8.3 and the other (pK = 9.28–9.69) in the pH range 8.5–9.9. It is pointed out that when the experimental conditions approached one another, there was agreement between the pK values obtained from titrimetric and kinetic studies. Evidence is presented from the literature concerning the validity of the pK value near 7.5 for the ε-amino groups in RNAse. The studies were repeated with performic acid oxidized RNAse and the 10 ε-amino groups were found to be monophasic with pK values between 8.01 and 8.10. The α-amino group of the N-terminal lysine was biphasic with a pK of 7.26 (pH range 7–8) and 8.13 (pH range 8.2–9.5).     

Chemical relaxation studies on bovine serum albumin. I. Reactions of the imidazole groups at pH 6 to 8

In the pH range of 6 to 8, the proton transfer reactions of BSA were measured using the temperature jump relaxation technique. The rate data were compared to the results using imidazole itself to establish that the observed reactions were those of the imidazole groups in the BSA. Upon the addition of calcium and gadolinium to the BSA solutions, no metal ion complexation was observed for either cation at the imidazole sites.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.