Ribonuclease viscosity

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). 

The initial decrease in optical rotation found in aqueous solutions of /3-lactoglobulin and ovalbumin is not, however, sufficient to differentiate globular proteins from simpler synthetic polypeptides in their transition behavior, for neither ribonuclease nor human serum albumin appear to exhibit it. The specific rotation of ribonuclease in water-2-chloroethanol mixtures becomes steadily less levorotatory as the proportion of nonpolar solvent increases (Weber and Tanford, 1959). In the case of human serum albumin Bresler (1958) and Bresler el al. (1959) find that only progre.ssive increases in specific rotation occur as the concentration of 2-chloroethanol is increased and that this change is accompanied by a steady rise in viscosity and the corresponding axial ratios characteristic of the formation of rodlike particles. If these proteins do have some initial helical content in water, as can be argued from their optical rotatory dispersion, then it appears that hydrophobic forces are not required for the stability of these regions. 

Most polypeptide chains devoid of cross-links assume a random-coil conformation in 8 M urea or 6 M guanidinium chloride, as evidenced by physical properties such as viscosity and optical activity. When ribonuclease was treated with P-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment (Figure 3.53). 

Tsong, T.Y. Baldwin, R.L. Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease a chain folding. Biopolymers 1978,17, 1669-1678. 

The diffusion coefficients D of macromolecules in dilute solutions have values of 10 cm /s (Table 7-1). The diffusion coefficients of the proteins ribonuclease, hemoglobin, collagen, and myosin, as well as deoxyribonuclease, were measured in dilute aqueous salt solutions. They were transformed into pure water diffusion coefficients by assuming that any differences are caused by differing viscosities only, and not by changes in the asymmetry or solvation coefficients. The diffusion coefficients of macromolecules in the melt are much lower, being 10" -10 cm /s. 

Analogous shifts in protein spectra have been observed as a result of conformational changes associated with denaturation. Initial reports on this phenomenon (see Ref 24) attributed the spectral difference to the transfer of the aromatie amino acids from the hydrophobic interior of the protein to the more aqueous surface environment as a result of the conformational change. Spectral changes for several proteins correlated well with independent measurements of denaturation such as intrinsic viscosity, circular dichroic spectra, and heat capacity measurements. For example, Edelhoch [26] compared the ribonuclease UV spectrum in buffer with that obtained in 6M guanidine hydrochloride (GuHCI). 

PDA analysis was also applied to the study of ribonuclease A in reversed-phase chromatography [33]. Ribonuclease A conformation has been the focus of numerous studies, and its dcnaturation is an excellent example of a reversible unfolding process. Cohen et al. [31] first reported the influence of a hydrophobic stationary phase on ribonuclease dcnaturation. In this work, the change in the 254/280 nm absorbance ratio was followed using multiple injections of the same sample. The increase in the ratio correlated extremely well with a number of other physical measurements of structural changes such as circular dichroism and intrinsic viscosity. 

For undetermined reasons, the optical density of DNA is less than the sum of the densities of the component nucleotides.Since DNA is a very long polymer, with a molecular weight of approximately 1,000,000, there is a decrease in viscosity on hydrolysis. The binding of basic dyes is characteristic only of large polymers, and has been used to assay hydrolysis. The most widely used assays, as with ribonuclease, measure either acid produced by hydrolysis of phosphate diesters or the formation of acid-soluble nucleotides. ... 

Figure 5.9 Viscosity changes by the action of heat on the enzyme ribonuclease...

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