Reduction and Carboxymethylation

Because cysteine residues are prone to autooxidation, proteins that contain disulfide bonds or free sulfhydryl groups must be subjected to reduction-alkylation process prior to cleavage. Alkylation converts sulfhydryl groups to stable derivatives. Also, during the reduction-alkylation procedure, the three-dimensional structure of proteins is disrupted to allow more cleavage sites to be accessible. Proteins are first reduced by treatment with 2-mercaptoethanol or dithiothreitol to convert disulfide bonds to free sulfhydryl group  

The reduction is carried out at alkahne pH (ca. 8.0) to generate a reactive intermediate (thiolate anion). 4-Vinylpyridine, iodoacetamide, or acrylamide can also be used in place of iodoacetic acid. 

Solution Reduction of a protein converts each sulfide bond to two sulfhydryl groups. Therefore, after reduction there are a total of2x2- -3 = 7 sulfhydryl groups in the protein. Because alkylation of each sulfhydryl group by iodoacetic acid increases the mass by 58 Da, the mass of the protein increases by 7 x 58 = 406 Da. 

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Scanu, A. M., The effect of reduction and carboxymethylation on the circular dichroic spectra of pure polypeptide classes of serum high density lipoproteins. BwMm. Biophys. Acta 200, 570-572 (1970). 

The stress-70 proteins interact with a broad spectrum of polypeptide substrates, but they have some degree of specificity in their interactions. In several instances, it has been shown that a stress-70 protein can bind to proteins [e.g., bovine pancreatic trypsin inhibitor (BPTI), a-lactalbumin] that have been stabilized in a nonnative, or denatured, form by reduction and carboxymethylation of the cysteines that would normally form disulfides at the same time, they will not bind to the native forms of the same proteins (Liberek et al., 1991b Palleros et ai, 1991, 1992). This suggests that the peptide-binding activity of the stress-70 proteins discriminates in favor of polypeptides in a denatured, and possibly extended, conformation over those in a compact secondary and tertiary structure. NMR experiments demonstrating that the E. coli dnaK... 

Subsequent studies showed that reduction and carboxymethylation of crystalline cytochrome 62 yielded two unlike subunits of approximately... 

In order to determine the positions of the Arg residues modified in the sequence of N. naja atra PLA2, PLA2 and the Arg-modified derivatives were digested with trypsin after reduction and carboxymethylation The tryptic... 

Reduction and carboxymethylation (or carboxamidomethylation) The following generally applicable procedure is based on the method of Crestfield et al. (1963), and has been widely used with minor variations for the past ten years. 

Reduction and Carboxymethylation of Proteins. Reduction of disulfide bridges with mercaptoethanol in 8M urea and reaction with iodo-acetate to introduce the carboxymethyl (CM) group onto the protein sulfhydryl groups have been described earlier (39). 

The complement-activating capacity of the Fc fragment of human IgG increases after selective reduction and carboxymethylation of inter-chain disulphide bonds. Differences observed in the c.d. spectra of the intact and modified fragment suggest that minor conformational differences exist between them. 

Reduction and carboxymethylation of A. niger glucoamylase resulted in complete loss of enzymic activity, whereas O-acetylation of three of the 13 tyrosyl residues resulted in loss of 20% of the enzymic activity. The tryptophanyl residues of Rhizopus niveus glucoamylase have been modified by treatment with A-bromosuccinimide so that the subsite structure of the enzyme could be studied. ... 

Photolytic. The UV photolysis (7, = 300 nm) of bifenox in various solvents was studied by Ruzo et al. (1980). In water, 2,4-dichloro-3 -(carboxymethyl)-4 -hydroxydiphenyl ether and 2,4-di-chloro-3 -(carboxymethyl)-4 -aminodiphenyl ether were identifled. In cyclohexane, 2,4-dichloro-4 -nitrodiphenyl ether and methyl formate were the major products. In methanol, a dichloro-methoxy phenol was identified. Photodegradation occurred via reductive dechlorination, de-carboxymethylation, nitro group reduction, and cleavage of the ether linkage (Ruzo et al., 1980). 

That the N-terminal methionine is exposed to the solvent was also concluded from studies directed toward the reduction and alkylation of the disulfides of rhGH. The conditions that were found to be optimal for the derivatization of the four sulfhydryls of rhGH resulted in the production of a side reaction when the procedure was applied to the methionyl analog. This side product was identified as containing carboxymethyl-S-methionine at the amino terminal residue. 

It is possible that racemization of some of the amiiio aeids, such as cystine, serine, and threonine, occurs during extraction and accounts for some of the complexity of wool protein fractions (Lindley, unpublished observations, 1962). Performic acid, however, used in the preparation of the keratoses did not produce racemization in proteins (Hill and Smith, 1957). It has not proved possible to solve uneciuivocally the problem of whether or not the reduction and alkylation pi oceduros used in the preparation of SCM kerateiues cause racemization. Lindley (unpublished, 1961) has shown that S -carboxymethyl cysteine isolated from acid hydrolyzates of SCM kerateines is partially racemized as measured both by direct optical rotation procedures and also by the use of a C-S lyase enzyme which is specihe for the n-form (Schwinuner and Kjaer, 1960). Control experiments showed, however, that L-S-carboxymethyl cysteine itself is partially racemized on refluxing with 5 N acid, and when allowance was made for this it appeared that the amount of racemization attributable to the reduction and alkylation procedures was small (less than 5 %) even when the most drastic conditions (pH 12.5 and 50°C) were used to prepare the SCM kerateines. Since S-carboxymethyl cysteine in peptide combination may well racemize more readily on acid hydrolysis than does the free amino acid, even this may be an over-estimate, and it would seem unlikely therefore that racemization is a serious problem in SCM kerateines as presently prepared. 

The protein is completely hydrolyzed by acid (6 N HCl, 24 hours or longer at 110°C, under vacuum or inert gas) to its constituent amino acids and the resultant hydrolysate is evaporated to dryness. The amino acid composition is determined on protein hydrolysates obtained after 24,48, and 72 hours of acid treatment. The content of amino acids with bulky aliphatic side chains such as isoleucine, leucine, and valine, which undergo slow hydrolysis, is calculated from an extrapolation of the hydrolysate data to infinite time. The content of hydroxyl-containing amino acids, which are slowly destroyed during hydrolysis, is obtained by a corresponding extrapolation to zero time. Since cysteine, cystine, and methionine residues are somewhat unstable to hydrolysis, these residues are oxidized to cysteic acid and methionine sulfone, respectively, with performic acid before quantitative analysis. Cysteine, or half-cystine, is quantitated as a derivative such as carboxymethyl cysteine after reduction and alkylation, a necessary prerequisite to subsequent sequence analysis. Tryptophan... 

Irradiated ribonuclease is treated with H235S and the non-covalently bound 35S is removed by Sephadex chromatography. At this stage the specific activity of the ribonuclease was 0.089 /xc/mg., of which 0.043 /xc/mg. was caused by /2-labeling determined in a separate experiment. Reduction of this material with mercaptoethanol in 8M urea, precipitation, and carboxymethylation resulted in ribonuclease s having a specific activity... 

Mb-CMC fihn was made by casting solution of myoglobin and carboxymethyl cellulose (CMC) on pyrolytic graphite electrode. Trichloroacetic acid (TCA), nitrite, oxygen, and hydrogen peroxide can be catalytically reduced at the Mb-CMC film electrode. When TCA was added to a pH 3.0 buffer, an increase in the MbFe reduction peak at about -0.2 V was observed, accompanied by a decrease of the MbFe oxidation peak. The reduction peak current increased as the TCA concentration increased. The catalytic reduction peak current showed a linear relationship with TCA concentration in the range of 7.5 X 10 - 2 X 10 M. Compared with the direct reduction... 

A second metallic contaminant of interest is Cr. Remediation of Cr-contaminated soils is centered around the reduction of Cr(VI), a more toxic and soluble form, to Cr(III), which is considerably less soluble and which readily precipitates out of solution. A number of different nanoparticles have been shown to possess this capability, including ferritin protein cages containing fenihydrite (Fe(O)OH), palladium (Pd) nanoparticles, and carboxymethyl cellulose (CMC)-stabilized Fe ) (87, 89, 93). In both the ferritin cages and the CMC-Fe p), the active species was a form of Fe, either Fe(0) in the case of the CMC-Fe p), or Fe(II) in the case of the ferritin cages. The reaction known to reduce Cr(VI) utilizing Fe(II) is shown in Equation (20.2). 

Complexes comprising HA-Na and carboxymethyl cellulose have been developed as gel-coats or membrane barriers for the reduction of postoperative adhesion formation (see also Chapter 8 for details). 

The carboxymethylation of horse heart cytochrome c by [2-i C] bromo-acetate has been monitored by NMR spectroscopy (77). In the absence of cyanide ion position 65 of the cytochrome c is the only reactive methionine. Carboxymethylation gives as expected a single resonance at 48.4 ppm (similar to carboxymethylated methionine peptides). In the presence of cyanide ion the methionines at positions 65 and 80 are both reactive and two resonances for the carboxymethylated product were observed 48.4 ppm position 65 48.2 ppm position 80. Upon reduction of the iron and carboxymethylation, the position 80 carboxymethylmethionine moves to 48.4 ppm, suggesting that the carboxymethylmethionine-80 feels the oxidation state of the metal (Fe +-Fe +). At long reaction times further carboxymethylations were detected. 

A modified (horse) cytochrome c, in which Met-80 is carboxymethylated and therefore unable to coordinate to Fe, was studied by Di Marino et al [145]. At pH 7 the Fe(II) form, by analogy with deoxymyoglobin, is able to bind Oj and CO. Thus the sixth coordination position is vacant or is occupied by weakly bound HjO. With a Au electrode, either as wire or coated onto carbon, but without further modification, cyclic voltammetry was observed with peaks due to reduction and oxidation. There was clear evidence for specific adsorption of oxidized reactant, and the authors did not analyze their results further. The reduction potential was determined to be — 218 mV from equilibrium measurements. 

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