The effect of a number of polar organic solvents, including aliphatic alcohols, ethers, ketones, and nitriles, on the renaturation kinetics and stability of acid-soluble calfskin collagen has been investigated. The solvents reduced the rate
The effects of mono- and poly-hydric alcohols in the presence of KCl on the intrinsic stability of collagen molecules in dilute acid solution were compared with corresponding solvent and salt effects on the increased stability of the aggregated molecules in salt-precipitated fibrils. Salt addition decreased solubility and increased the thermal stability of fibrils, but progressively decreased the stability of collagen molecules in solution. In contrast, the alcohols enhanced solubility and decreased fibril stability, the effects increasing with solvent hydrocarbon chain length and with decreasing hydroxyl/methylene-group ratio. Molar destabilization of dissolved collagen by alcohols was lower than for fibrils, and at low salt concentration, both ethylene glycol and glycerol were structural stabilizers. Electron-micrograph studies indicated that salt-precipitated fibrils tended to adopt the native aggregation mode, and qualitatively similar solvent effects were observed in insoluble collagens. Implications of the experimental findings are discussed in terms of a model in which electrostatic and apolar interactions mainly govern the excess of stability in collagen fibrils whereas intrinsic stability of single molecules is a function of polar interactions and polypeptide-chain rigidity.
The effects of a number of related diols, substituted diols and glycerol on the thermal stability of acid-soluble calf skin collagen were investigated. Thermal transition temperatures were determined by optical rotation measurement. Short-chain diols with terminal hydroxyl groups, i.e. ethylene glycol and propane-1,3-diol, stabilized the protein at all accessible concentrations. Stabilization was also observed with glycerol and diethylene glycol. Higher homologues in the diol series produced various effects, as did hydroxyl-group positional isomerism. Monoalkyl substitution of diols progressively lowered the denaturation temperature of collagen. Results are discussed in relation to possible mechanisms of perturbant action.
The lyotropic effect of urea and certain neutral salts on protein structures in solution is well established and is generally evident in a lowering of the characteristic thermal transition temperature at which the native ordered conformation is transformed into a random-coil form (Steven & Tristram, 1962;Hippel & Wong, 1963; Tanford, 1967). In the present communication, preliminary results obtained when solutions of acid-soluble calf-skin collagen were renatured in the presence of urea are reported. These studies suggest that at moderate urea concentrations an appreciable increase in the rate and extent of reversion as measured by physicochemical techniques is effected.Renaturation kinetics were followed by opticalrotation and reduced-viscosity measurements at 15°by using procedures described by Davidson & Cooper (1967). Acid-soluble calf-skin collagen (Cooper & Davidson, 1965) in 0-15M-potassium acetate buffer, pH4-8, was denatured at 450 for 15min. before addition of the required amounts of urea and dilution to volume with buffer to give a final concentration of 0-86mg. of dry protein/ml.Increasing urea concentration progressively decreased the initial rate of back mutarotation (Fig. la), although the rates of the subsequent slow mutarotation after 2hr. were greater than that of the control. Thus the reaction curve with 0.5 M-urea crossed that of the control after approx. 8hr., giving reversion values after 32 hr. slightly in excess of the control. It was apparent that a similar situation would arise at higher urea concentrations if the renaturation periods were to be extended. Although there was evidence of progressive decrease in the initial rate of reduced-viscosity recovery with increasing urea concentration similar to that noted in the mutarotation reaction profiles, this effect was largely obscured by the subsequent increase in the rate and extent of recovery in urea concentrations up to 2-0M, with maximum effect at about 1-OM (Fig. 1c). The optimum regain noted with 1-OMurea was particularly striking, since, in terms of optical-rotation recovery, reversion after 32hr. was less than that of the control. The effect of urea concentration on the 32 hr. optical-rotation and reduced-viscosity recovery values is shown in Figs. 1(b) and 1(d), from which the occurrence of an optimum concentration range in each case is apparent.
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