Control of aggregation in protein refolding: the temperature-leap tactic.

The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4 degrees to 36 degrees, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20 degrees (approximately 60%), is greater than that at either 4 degrees or 36 degrees. However, if refolding is conducted at 4 degrees, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36 degrees, the yield rises rapidly to 95% at 150 min. This is an example of the "temperature leap" tactic. These results can be understood on the basis of two slow-folding intermediate whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4 degrees, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a "leap" to 36 degrees for 30 additional minutes. With initial incubations from 4 degrees to 12 degrees, the final yield is > 90%, drops rapidly from 12 degrees to 20 degrees, and decreases more gradually to approximately 45% at 36 degrees. The overall results qualitatively fit the simple idea of ordinary temperature-accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature-leap approach to find application in refolding other proteins.

Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II.

The denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of approximately 0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle-forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.

Nucleation in protein folding--confusion of structure and process.

The term 'nucleation' is currently used to denote two distinctly different aspects of folding: the kinetic and the structural. This gives rise to ambiguity in the use of the word 'nucleation', which is compounded by the fact that the word 'nuclei', as used in the structural sense, has more aliases than cats have lives.

Surfactant-mediated hydrophobic interaction chromatography of proteins: gradient elution.

Addition of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate (CHAPS) to mobile phases in gradient elution hydrophobic interaction chromatography (HIC) on SynChropak Propyl causes changes in observed elution times for nine globular proteins. The nine proteins showed different percentage reductions in capacity factor, k', demonstrating the ability of CHAPS to change the selectivity of the separations. Three basic types of gradient experiments have been explored for surfactant-mediated gradient elution HIC. Type I gradients are conducted with constant salt and variable surfactant concentration. Type II gradients with variable salt and constant surfactant concentration, and Type III gradients with variable salt and surfactant concentrations. By the criterion of a linear relationship between gradient time and retention time the linear solvent strength condition applies to Type II and Type III gradients. Type III gradients, with the fastest re-equilibration time, are preferable for repetitive analyses. Type I gradients are relatively ineffective in making use of the solvent strength of CHAPS, and Types I and II gradients require long equilibration times due to large changes in surface concentration of CHAPS which occur during elution. The presence of CHAPS had a negligible effect on peak shapes of the proteins examined, except for bovine serum albumin which yielded a narrower, less distorted peak in the presence of CHAPS.

Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins.

Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.

The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential.

The rate and yield of oxidative renaturation of reduced RNase A has been studied as a function of [-S-S-]/[-SH]. The principal conclusion of these studies is that rates and yields of oxidative renaturation are strongly dependent on the low mol. wt disulfide/thiol ratio. The relationships are complex and do not parallel the redox potential of the system. The present results are consistent with earlier findings on other proteins, and lead us to believe that the above conclusion is general. Kinetic studies of oxidative renaturation should recognize and account for the dependence of reaction rate and extent on the disulfide/thiol ratio. This ratio can change substantially over the course of a reaction, either due to stoichiometric transfer of disulfide to protein, and/or adventitious air oxidation of thiols. Failure to account for changes in the disulfide/thiol ratio may compromise the interpretation of such experiments.

Surfactant-mediated protein hydrophobic-interaction chromatography.

Three proteins have been subjected to hydrophobic-interaction chromatography in the presence of submicellar concentrations of the surfactant (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). At several concentrations of CHAPS below the critical micelle concentration, CHAPS increased the retention of lysozyme and pancreatic trypsin inhibitor, but decreased that of ribonuclease A. The dependence of retention on CHAPS concentration was substantially different for the three proteins, i.e., the surfactant showed selectivity in its interactions with the proteins. In the solvents that were used for chromatography the surface tension decreases monotonically with increasing CHAPS concentration. Since different proteins may either be eluted or retained by the addition of CHAPS, our findings are inconsistent with the idea that retention is a simple function of surface tension. It also appears unlikely that the selectivity we have observed can be accounted for by a formulation of retention as a function of surface tension. As an alternative interpretation, we present a scheme of multiple equilibria and their kinetic components as a basis for formulating the dependence of retention on surfactant concentration.

Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins. Theory and experiment.

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k' = m log (1/[Ca2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log-log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k' values at or near the column inlet was observed.

Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme.

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.

Effects of urea-thermal denaturation on the high-performance cation-exchange chromatography of alpha-chymotrypsinogen-A.

Retention parameters of alpha-chymotrypsinogen-A were determined by isocratic elution for a series of concentrations of calcium acetate and sodium acetate both in the presence and absence of urea. Under non-denaturing conditions of temperature and urea concentration, urea facilitated elution. Under reversible denaturing conditions a sharp drop in chromatographic retention was observed over a narrow temperature range which could be correlated with equilibrium measurements of the protein fluorescence. Retention of both native and denatured protein could be fit to a non-mechanistic retention model by plotting the log k' against log salt concentration. Conventional interpretation of these findings indicates that, while the number of ions displaced during binding is greater for the denatured protein, the affinity per ion decreases since the retention of denatured protein is much less than native. Elution profiles obtained under partially denaturing conditions showed a strong flow-rate dependence. We attribute these observations to a rate of equilibration between native and denatured protein that is on the timescale of the chromatographic rate processes.

Influence of urea on the high-performance cation-exchange chromatography of hen egg white lysozyme.

The effects of urea on the high-performance cation-exchange chromatography of hen egg lysozyme are reported. The capacity factor, k', has been determined as a function of cation concentration with a polyaspartate column using the acetates of Na+, K+, Ca2+ and Mg2+. Urea decreases lysozyme retention. Plots of log k' vs. log ionic strength show linear relationships. The slope of the plot describing the Ca2+ elution of lysozyme was the same in the presence of 5 M urea as in its absence. In strong urea solutions and at elevated temperatures, lysozyme denaturation is evidenced by a marked decrease in k'. The temperature range for denaturation corresponded closely to that observed in intrinsic fluorescence and circular dichroism measurements. The potential utility and limitations of high-performance ion-exchange chromatography for studying protein denaturation are discussed.

Multiple parameter kinetic studies of the oxidative folding of reduced lysozyme.

We have compared the oxidative renaturation of reduced hen egg white lysozyme promoted by Cu(II) + O2 with that promoted by a glutathione redox buffer. The progress curves for protein fluorescence, circular dicroism, thiol oxidation, hydrodynamic volume, and enzymic activity were determined for both regeneration systems. All of these processes were more rapid in the glutathione regeneration than in the copper-catalyzed. Comparison of the two systems was carried out by normalizing the progress curves with a coordinate system where "time" is replaced by "extent of protein thiol oxidation." While similar progress curves were obtained for circular dichroism, the two systems produced distinctly different progress curves for enzymic activity, fluorescence, and gel permeation chromatographic reflection of protein hydrodynamic volume. We infer that all these differences result from differences in relative amounts and/or kind of reaction intermediates. Thus, there are substantial differences between the renaturation mechanisms of the glutathione- and the copper-promoted systems.