Ionic strength ribonuclease

Fig. 6. Plateau-values, I"P1 /mg m 2, of adsorption isotherms of lysozyme (LSZ), ribonuclease (RNase), a -lactalbumin (aLA), calcium-depleted (X -lactalbumin (aLA(-Ca )) and bovine serum albumin (BSA) on hydrophobic polystyrene (PS) and hydrophilic hematite (a — Fe203) and silica (Si02) surfaces. An indication of the charge density of the surface is given by the zeta-potential, C, and of the proteins by + and signs. Ionic strength 0.05 M T = 25°C. (Derived from Currie et al. 2003).

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). 

FIGURE 6 Effect of ionic strength on protein solubilization in AOT-iso-octane system with no pH control (O) cytochrome c, ( ) lysozyme, (a) ribonuclease-a (from Goklen and Hatton49 by courtesy of Marcel Dekker, Inc.). 

Fig. 11. Dissociation of the phenolic groups of ribonuclease at ionic strength 0.15. The dashed lines show regions of time-dependence. Half-filled circles represent measurements after reversal from pH 11.5 (middle curve) and after reversal from pH 12.7 (upper curve). O—T = 25°C —T = O C. From Tanford el al. (1955a).

Figure 13 shows the data for the three phenolic groups of ribonuclease which ionize reversibly (Tanford etal., 1955a), based on spectrophotometric titration curves such as Fig. 11. A straight-line plot is obtained, in agreement with Eq. (14). The values of w are 0.112, 0.093, and 0.061, respectively, at ionic strengths 0.01, 0.03, and 0.15. (The salt used to produce the ionic strength was KCl, and there is evidence that neither K" nor CF is bound to an appreciable extent. The use of Zn as abscissa is therefore presumably acceptable.) Comparison with the calculated values of Table III shows that the experimental values are lower than predicted by about 20%. Such a deviation must be considered almost within the error of calculation. [If the radius of the hydrodynamically equivalent sphere (19 A) had been used as the basis of calculation, the calculated values of w would have become 0.119, 0.096, and 0.066, respectively.]...

Fig. If). The change in the difference spectrum of 1.8 X M ribonuclease with time in 7 M urea, 0.04 ionic strength imidazole, pH 7.0 at 23 -24°C. Each spectrum was scanned starting at the time shown at 300 in and was completed at 250 mu 50 sec later. Each division on the vertical axis represents 0.1 absorbancy unit. Values above each baseline represent increased absorbancy of ribonuclease in urea over that in its absence. (Nelson and Hummel, 1962.)...

Breslow R, Dong SD, Webb Y, Xu R. Further studies on the buffer-catalyzed cleavage and isomerization of uridyluridine. Medium and ionic strength effects on catalysis by morpholine, imidazole, and acetate buffers help clarify the mechanisms involved and their relationship to the mechanism used by the enzyme ribonuclease and by a ribonuclease mimic. J. Am. Chem. Soc. 1996 118 6588-6600. 

Eftink, M. R. and Biltonen, R. L. (1983) Energetics of Ribonuclease A Catalysis. 1. pH, Ionic Strength, and Solvent Isotope Dependence of the Hydrolysis of Cytidine Cyclic 2, 3 Phosphate, Biochemistry 22,... 

Figure 4. Effect of ionic strength on solubilization of three proteins in AOT system, (o) cytochrome c ( ) lysozyme (A) ribonuclease-A. (Reproduced with permission from Ref. 38. Copyright 1987 M. Dekker.)...

Figure 3. Phase boundaries (r = 100) of six proteins, plotted as net surface charge density (net charge/nm ) vs. 1 (ionic strength) (O) bovine serum albumin ( ) lysozyme (A) ribonuclease (A) chicken egg albumin ( ) -lactoglobulin and ( ) trypsin.

The tertiary structure describes the complete three-dimensional stmcture of the whole polypeptide chain. It includes the relationship of different domains formed by the protein s secondary structure and the interactions of the amino acid substituent -R groups. An example of a protein chain with a-helices and /3-folding, the enzyme ribonuclease, is shown in Fig. 1.17. The specific folding of a protein is only thermodynamically stable within a restricted range of environmental parameters, i.e. the right temperature, pH and ionic strength. Outside of this range, the protein could unfold and lose its activity. 

Fig. 164. Optical densities of solutions of ribonuclease at 1.90 mg. per milliliter, ionic strength 0.16, as a function of temperature, when compared with a reference solution of zero optical density at pH 6.83 and low temperature (<43 ). The pH s of the solutions are indicated (Hermans and Scheraga, 1961a).

The association of bamase, an extracellular ribonuclease, with its intracellular inhibitor, barstar, provides a particularly well-characterized example of electrostatically steered protein-protein encounter. The association rate is very fast (about 10 -10 at 50 mM ionic strength), and mutation and ionic-strength-dependence studies clearly show the influence of electrostatic interactions. Brownian dynamics simulations are able to reproduce the ionic-strength dependence of the rate for the wild-type proteins and the rates for wild-type and 11 mutants at 50 mM ionic strength to within a factor of 2. These simulations provide insight into the structure of the encounter (transition state) complex in which barstar tends to be shifted from its position in the bound complex towards the guanine binding loop on bamase. 

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