Enhanced collagen renaturation in the presence of a lyotropic agent

Abstract

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.

Effect of compounds of the urea–guanidinium class on renaturation and thermal stability of acid-soluble collagen

“…Thus in the viscosity-reversion profiles, which were particularly sensitive to the slow reversion process, recoveries after 1 h at 0.25Mand 0.5M-guanidinium bromide exceeded that of the control. Similar effects were observed previously in collagen renaturation profiles obtained at various urea concentrations (Russell & Cooper, 1969b). Changes in initial kinetic characteristics were confirmed in the present study by deviation of the experimental curves from second-order kinetics after periods corresponding to two to three reaction halflives.…”

“…The results of formamide and urea addition to a renaturing acid-soluble collagen system and effects on thermal stability have been described in previous reports (Russell & Cooper, 1969b. In the present study, this approach has been extended to compare the effects of a range of guanidinium salts with those of formamide and urea.…”

“…As with these uncharged amides, recovery maxima of similar height were observed at intermediate guanidinium Table 1. Molar activities of amides, guanidinium and potassium salts in renaturation Values for formamide and urea were calculated from data reported by Russell & Cooper (1969b Values for the collagen in 0.15M-potassium acetate buffer are given as a function of amide and guanidinium salt concentration. Symbols and explanation are as given for Figs.…”

Mixed ligand complexes of toxic metal ions with L-glutamic acid and L-methionine in urea-water mixtures

“…Effect of urea Urea acts as a denaturant of DNA (Vonhippel and Schleich, 1968), proteins (Russel and Cooper, 1969), insulin (Thurow, 1979) and other macromolecules (Siezen and Bindels, 1982) but this action is reversible. Urea molecules exhibit their influence through changes in water structure (Gordon and Warren, 1968).…”

The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment‐long‐spacing aggregates from collagen solution

“…Many chemical agents, some of which are endogenous to the extracellular milieu, affect the rate of fibrillogenesis. These include glycosaminoglycans [9-161, glycoproteins [17, 181, citrate [19], ascorbic acid [20], neutral salts [21], DNA and RNA 19,221, urea [23,24], and catecholamines [25]. Nonbiological substances have also been shown to alter the rate of collagen fibrillogenesis in vitro.…”