Ribonuclease, oxidation

Mandel, M. Proton Magnetic Resonance Spectra of Some Proteins. I. Ribonuclease, Oxidized Ribonuclease, Lysozyme and Cytochrome C. J. Biol. Chem. 240, 1586-1592 (1965). 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Qi et al. (1998) have demonstrated that ribonuclease A exhibits behavior like that of cytochrome c. The burst phase observed on dilution of Gdm HCl-denatured RNase A is mimicked exactly by reduced RNase A. The latter, when carboxamidomethylated to prevent oxidation, has a CD at 222 nm that is nearly independent of temperature and indicative of extensive unfolding at zero denaturant. 

Vanadium is beneficial and possibly essential for humans. It is certainly essential for a number of organisms. Vanadate (oxidation state V) and its derivatives are phosphate analogues, showing both ground state and transition state analogy (both structural and electronic) with phosphorus compounds. The analogy of five-coordinate vanadium compounds with the transition state of phosphate ester hydrolysis is well documented, and explains why so many vanadium compounds are potent inhibitors of phosphatases, ribonucleases and ATPases. 

E. Haas, G. T. Montelione, C. A. McWherter, and H. A. Scheraga, Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements, Biochemistry 26, 1672-1683 (1987). 

Among the specific enzymes whose activity has been reported to be decreased after in vitro ozone exposure are papain, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, ribonuclease, and acetylcholinesterase. The latter enzyme appears to be particulady susceptible to free-radical and oxidative states. A loss in acetylcholinesterase activity has been reported in the red cells of humans and mice that inhaled ozone. However, there are only minimal amounts of this enzyme in lupg tissue, and, although it has been suggested that acetylcholinesterase is important in bronchial tract ciliary activity, there is no direct evidence to support this conjecture. 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

Oxidation of mono-cysteine peptides to the dimer is a straightforward reaction that can produce only the desired product. In the case of bis-cysteine peptides statistically the oxidation leads to the homodimers in parallel and antiparallel orientation as well as to the disulfide-bridged monomer and oligomers. When the two cysteine residues are placed in the adjacent position formation of homodimers is highly favored over the cyclic monomer (Section 6.1.5.1) and the product distribution depends strongly on the peptide concentration. Such a type of intermolecular disulfide bridging is present in bovine seminal ribonuclease, where an antiparallel alignment occurs at the interface of the dimer. 97 ... 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

SO is capable of oxidizing reduced ribonuclease and restoring enzymic activity, suggesting that its physiological role may be the non-random formation of protein disulphide bonds, e.g. during protein biosynthesis. 

The intrinsic viscosity of native ribonuclease is very low. Harrington and Schellman (247) reported 3.3 ml/g at neutral pH in 0.1 M KC1. Buzzell and Tanford (265) found values of 3.3-3.5 ml/g over the entire pH range from 1 to 11 and ionic strengths from 0.05 to 0.25 M. This value increases dramatically on denaturation even without oxidation or reduction of the disulfide bonds to 8.5 ml/g (266). In the presence of reducing agents and 6 M guanidine hydrochloride the value is 16.0 ml/g (267). 

Prutz WA, Butler J, Land EJ (1985a) Methionyl —> tyrosyl radical transitions initiated by Br2 in peptide model systems and ribonuclease A. Int J Radiat Biol 47 149-156 Prutz WA, Monig H, Butler J, Land E (1985b) Reactions of nitrogen dioxide in aqueous model systems oxidation of tyrosine units in peptides and proteins. Arch Biochem Biophys 243 125-134 Redpath JL, Willson RL (1975) Chain reactions and radiosensitization model enzyme studies. Int J Radiat Biol 27 389-398... 

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