Guanidine hydrochloride, oxidation

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). 

Disulfide linkages may be broken either oxidatively or reductively. The former method involves the treatment of the protein with performic add, which converts all disulfide bonds into cysteic add residues. This procedure is usually performed before a protein is hydrolyzed for amino add analysis. Cystine and cysteine are then determined as cysteic add. The reductive cleavage of disulfide bonds involves the treatment of the protein with mercaptoethanol (SH-CH2-CH2-OH), followed by the alkylation of the newly formed -SH groups. The complete disruption of all secondary interactions (that is, complete denaturation) can be achieved in most proteins with 6 M guanidine hydrochloride and 0.1 M mercaptoethanol or 8 M urea and 0.1 M mercaptoethanol. 

For their study Tanford et al. have chosen aqueous 6 M guanidine hydrochloride - -0.1 M mercaptoethanol (to rupture disulfide bonds and to prevent their formation by oxidation of tliiol groups) as denaturing solvent medium. As the initial and most critical test of random coil behaviour, hydrodynamic measurements were carried out on a number of proteins dissolved in the above-mendioned medium. 

Fig. 2. Reverse-phase HPLC profile of r-hGMF-beta. (A) Pure r-hGMF-beta without treatment. (B) After treatment with 10 mM DTT in presence of 6M guanidine hydrochloride. Note identical retention time in each case. (C D) Time course of oxidative refolding of r-hGMF-beta and separation of isoforms by reverse-phase HPLC. Pure r-hGMF-beta was dissolved in O.IM sodium phosphate and incubated at room temperature with reduced and oxidized glutathione in the presence of guanidine hydrochloride, as described in the text, for 4 h (C) and 8 h (D). (Adapted from ref. 17)...

It should be noted that low concentrations of denaturant can also favor protein reactivation. For example, incubation in low concentrations of denaturant was observed to promote proper refolding of chymo psinogen. The refolding of this protein from 6M guanidine hydrochloride is optimized by diluting to 1.2M GuHCl in the presence of reduced and oxidized glutathione (72). 

Recombinant proteins expressed at high levels in bacterial hosts are often found in the form of inclusion bodies (f). These inclusion bodies consist of dense masses of partially folded, reduced protein. In this state, the target proteins are inactive the inclusion bodies must be dissolved and the soluble protein must be refolded and oxidized into the native, active state. The typical downstream process for recovering protein from inclusion bodies includes two distinct operations the dissolution of the inclusion bodies at high concentrations of denaturant such as urea or guanidine hydrochloride followed by a dilution or gradual removal of the denaturant to permit folding and oxidation to occur ( - ). 

NH4OH gave 9. Oxidation of 9 with cupric acetate afforded 5-deoxy-L-arabinosone 10, followed by reaction with acetone oxime and 5% NH4OH to produce the a-keto aldoxime 11. Reaction of 11 with EtOCOCH(NH2)CN in ethanol afforded 12, which was treated with sodium methoxide and guanidine hydrochloride to afford 13. Reduction with sodium dithionite in aqueous solution buffered to pH 7 gave L-eryt/iro-biopterin (2). 

Dimethyl, methyl (polyethylene oxide acetate-capped) siloxane Disodium lauriminodipropionate Polysorbate 80 antistat, textile finishes Cetrimonium chloride Polyglyceryl-10 tetraoleate Stearyl hydroxyethyl imidazoline antistat, textile finishing Guanidine hydrochloride Quaternium-27 antistat, textile lubricants PEG-3 dioctoate PEG-2 oleate TEA-phosphate antistat, textile maintenance Quaternium-27 antistat, textile processing PEG castor oil antistat, textile scours Oleth-4 phosphate antistat, textile softeners Lauramine oxide antistat, textile spin finishes Alkyl trimethyl ammonium chloride Ceteareth Cetethyl morpholinium ethosulfate Cetoleth Cetrimonium bromide Lauramine oxide PEG cocamine PEG-2 oleamine PEG oleate PEG stearamine PEG stearate Polyethylene wax Polysorbate 60 Sodium cocoamphoacetate Trideceth phosphate... 

Ferric oxide Guanidine hydrochloride Heparin ammonium Heparin lithium Hexane Hydrochloric acid Hydrocinnamic alcohol Hydrocinnamyl acetate Hydrogen sulfide Isophthaloyl dichloride Manganese dioxide a-Methylbenzyl alcohol Myrtrimonium bromide Nickel oxide (ic) Oxalic acid dihydrate o-Phenetidine Phenolsulfonic acid Phenoxyacetic acid Phenylacetaldehyde Phenylacetaldehyde dimethyl acetal Phenyl acetate Phenylacetic acid ... 

Narayanasami R, Nishimura JS, McMillan K, Roman LJ, SheaTM, RobidaAM, Horowitz PM, Masters BS (1997) The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins. Nitric oxide Biol Chem 1 39-49... 

The equilibrium unfolding of the oxidized form of Escherichia coli thioredoxin at pH 7 was studied by Santoro and Bolen [1] using guanidine hydrochloride (GdnHCl) and urea as denaturant at 25°C by monitoring the changes in ellipticity at 222nm. 

The Os(VIII)-catalysed alkaline oxidation of guanidine hydrochloride by 104 ions is of fractional order in OH and zero order in substrate. The rate law for Mn(n)-catalysed oxidation of 2-amino-meto-xylene to 2,6-dimethyl-pura-benzoquinone in acetone-water solution has a complex dependence in H+ the rates decrease on increasing the ionic strength and decreasing the dielectric constant of the medium. The oxidation of myo-inositol has been studied in both alkaline and acidic solution and found to exhibit inverse fractional order in H" " and OH ions the alkaline reaction has an inverse fractional order in the substrate, whereas a fractional order is observed in the acidic reaction. The Ru(III)-catalysed oxidation of 2-methylcyclohexanol (mch) to 2-methylcyclohexan one in HCIO4 is zero order in mch and H" " ions. The rate of oxidation of L-serine to 2-hydroxyethanal in alkaline medium is retarded by OH ions and the ionic strength. ... 

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