High Denaturant Concentrations Research Articles

Stability of the larvicidal activity of Bacillus thuringiensis subsp. israelensis: amino acid modification and denaturants.

The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride.

Conformation and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC).

Spectrophotometric measurement was found to be a sensitive method for evaluating the stability of the chymotrypsin inhibitor from the winged bean. The thermal stability of this protein in aqueous solution was much greater at pH 3 than at pH 8 or pH 11. Evidence from u.v. absorption and from circular dichroism indicated that irreversible conformation changes occurred at higher temperature (greater than 70 degrees). Circular dichroism and optical rotatory dispersion studies at pH 8 show that the inhibitor is rich in beta-structure and virtually devoid of alpha-helix in aqueous solution. We conclude from experiments with denaturing solvents that the inhibitor is very stable and that high concentrations of denaturant are required before unfolding occurs. Chemical modification experiments with tetranitromethane were consistent with a tight stable structure; even in 6M guanidine hydrochloride only three of the five tyrosine residues in the inhibitor molecule were nitrated. However, tyrosine does not seem to be implicated at the reactive site of the inhibitor. Interaction of the inhibitor with alpha-chymotrypsin and chymotrypsin B was also followed by difference spectroscopy in the ultraviolet region. Difference spectra were detected that were characteristic of changes in the environment of both tyrosine and tryptophan chromophores. Comparison of the spectral data obtained for the interaction of the inhibitor with bovine alpha-chymotrypsin and with chymotrypsin B indicated that a tryptophan residue may be involved at the reactive site of the inhibitor. Spectral changes were also detected for the interaction between the chymotrypsin inhibitor and trypsin, although it is well established that the specificity of this inhibitor is restricted to the chymotrypsins.(ABSTRACT TRUNCATED AT 250 WORDS)

Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state.

Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.

Effect of sodium dodecyl sulphate on the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris)

The effect of sodium dodecyl sulphate on mustard and rapeseed 12S protein has been monitored by the techniques of ultracentrifugation, viscosity, difference spectra and fluorescence spectrophotometry. At low concentration of sodium dodecyl sulphate (<3.47 mM) mustard protein undergoes aggregation and at higher concentrations it dissociates to 1.8 S protein, the dissociation being complete at 17.3 mM sodium dodecyl sulphate. The rapeseed protein, on the other hand, undergoes dissociation at all the concentrations of sodium dodecyl sulphate. The reduced viscosity values of mustard protein in the presence of the denaturant are higher than those of rapeseed protein. Similarly in difference spectra change in absorbance values of mustard protein are higher.’ The relative fluorescence intensity of the mustard protein increases with sodium dodecyl sulphate concentration, upto 0.87 mM and this is followed by fluorescence quneching at higher denaturant concentrations. However, with the rapeseed protein fluorescence quenching was observed at all concentrations of sodium dodecyl sulphate.

Heme and cysteine microenvironments of tuna apomyoglobin. Evidence of two independent unfolding regions.

The heme and cysteine microenvironments of bluefin tuna apomyoglobin have been investigated by examining the fluorescence properties of two extrinsic chromophores, i.e., ANS and 1,5-AEDANS. 1,5-AEDANS was covalently bound to the single cysteine residue found in the primary structure of tuna apomyoglobin. Recombination experiments with hemin showed that tuna apomyoglobin does not bind 1,5-AEDANS in the same binding site of the heme, although the fluorescence properties of the covalently bound 1,5-AEDANS strongly suggest that the dye is embedded in a rather nonpolar microenvironment. ANS was selected because of its ability to bind the apomyoglobin in the same nonpolar moiety of the heme. Acidification of apoMb--AEDANS to pH 3.0 produced an increase of 1,5-AEDANS fluorescence intensity and a shift of its emission maximum from 475 to 470 nm. In the same pH range apomyoglobin lost its ability to bind ANS. Two independent transitions were observed with increasing concentrations of guanidine. Low guanidine concentration (less than 1.0 M) unfolded the heme binding site as indicated by the disappearance of ANS fluorescence, whereas higher denaturant concentration was required to produce full normalization of 1,5-AEDANS emission spectrum.

Dimeric intermediates in the dissociation of lactic dehydrogenase.

Rate-determining second-order reactions in the reconstitution of a number of oligomeric enzymes have shown that catalytic activity requires association. In order to answer the question whether in the case of tetrameric enzymes, e.g. lactic dehydrogenase, the bimolecular step belongs to the dimerization of the monomer or the dimer, attempts have been made to isolate intermediates of dissociation, and to study their reconstitution to the native tetramer. Varying concentrations of urea or guanidine · HCl were applied in order to labilize the native quaternary structure; incubation time, temperature and enzyme concentration were changed as additional variables. Both isoenzymes, M4 from pig and bovine skeletal muscle, and H4 from pig heart show separable steps in their denaturation profiles on changing the concentration of urea or guanidine · HCI. At high denaturant concentrations, denatured and deactivated subunits are formed. At about 2 M urea or 0.8–1.2 M guanidine · HCI (t≦ 5° C) metastable intermediates are obtained which represent inactive dimers, as shown by sedimentation analysis. Increased temperature yields wrong aggregates rather than low-molecular-weight intermediates, causing a drastic decrease of reactivation. Reconstitution of the metastable dimeric intermediates to reestablish the native tetramer is characterized by the same rate constants which have been reported previously for the overall reconstitution, starting from denatured monomers [R. Jaenicke (1979) FEBS Symp. 52, 187–198]. In the case of the isoenzyme M4 from pig skeletal muscle (after incubation in 1 M guanidine · HCl) reconstitution follows a simple bimolecular mechanism, while in the case of H4 from pig heart sigmoidal reactivation profiles indicate a more complicated mechanism involving rate-determining first-order processes. The fact there is no significant difference between the kinetics of reactivation during the dimer tetramer transition on the one hand, and the monomer tetramer overall reconstitution on the other, strongly suggests that the rate-determining step in the consecutive association reaction underlying reactivation as well as tetramer formation must be the dimerization of inactive dimers.

Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.

Renaturation of yeast inorganic pyrophosphatase denatured in urea and guanidine hydrochloride.

The renaturation of yeast inorganic pyrophosphatase [EC 3.6.1.1] (PPiase) denatured in guanidine-HCl and urea was studied. The molecular weight of PPiase was estimated to be ca. 63,000-70,000 by means of Sephadex G-75 column chromatography in 8 M urea and 6 M guanidine-HCl and by electrophoresis on polyacrylamide gel containing 8 M urea. The activities of PPiase denatured in various concentrations of denaturants were measured in the presence and absence of the denaturants. In the presence of the denaturants, enzymatic activity decreased as the denaturant concentration increased up to 1.5 M guanidine-HCl and 4 7 urea. The activities of PPiase denatured in these denaturants were not restored by dilution with buffer. However, the enzymatic activities of PPiase denatured at concentrations higher than 1.5 M guanidine-HCl and 4 M urea were restored by dilution with Tris-HCl buffer (pH 7.5). The recovery of the enzymatic activities of PPiase denatured in 3 to 6 M guanidine-HCl and 6 to 8 M urea was to a level of about 90% of the native enzyme. Irreversible denaturation of PPiase in lower denaturant concentrations was prevented in the presence of sulfhydryl reagents, dithiothreitol, glutathione, and 2-mercaptoethanol. In irreversibly denatured PPiase, the amount of free SH groups decreased markedly. These results indicated that in lower denaturant concentrations, SH groups in PPiase are very oxidizable and their oxidation may cause irreversible denaturation. In higher denaturant concentrations where PPiase was denatured completely, the SH groups became less reactive. The conformations of renatured PPiases was investigated by means of N-bromosuccinimide oxidation, fluorescence emission spectra and circular dichroism spectra. The PPiase denatured in 6 M guanidine-HCl showed fully restored native conformation, as checked by these methods, although renatured PPiase gave a trough in the 280 nm region of slightly less magnitude than that of PPiase. On the other hand, PPiase denatured in 8 M urea showed restored enzymatic activity, but restoration of its conformation was incomplete as compared to PPiase denatured in 6 M guanidine-HCl. PPiase renatured from material denatured in lower denaturant concentrations, such as 4 M urea and 1.5 M guanidine-HCl, had quite a different conformation from the native enzyme as judged from CD spectra, N-bromosuccinimide oxidation and fluorescence spectra. Differences in PPiases denatured in urea and guanidine-HCl were discussed in connection with the possible modification of amino groups in PPiase by cyanate ions.

Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium.

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

Kinetics of unfolding and refolding of proteins: III. Results for lysozyme

The kinetics of the reversible denaturation of lysozyme by guanidine hydrochloride have been studied over a wide range of pH and denaturant concentration. The results contrast sharply with those observed for cytochrome c. The interconversion of native (N) and denatured (D) states is strictly first-order in both directions under most conditions, showing that no intermediate forms of any kind accumulate to a significant extent during the reaction. At one pH (pH 2.6) experiments were extended to extreme denaturant concentrations, and under these conditions kinetic intermediates were observed. Analysis by the procedures of the first paper of this series Ikai & Tanford 1973 showed that the principal intermediate observed at low denaturant concentration must be different from that observed at high denaturant concentration, and that both must be on the direct pathway from N to D. This suggests that the over-all reaction mechanism is of the form ▪ and this mechanism is able to account for all of the observed results. The intermediate X 1 which accumulates transiently in the renaturation reaction at low concentrations of guanidine hydrochloride is spectrally very similar to the native state, i.e. it is probably a highly ordered state, but most of the interactions between surface acidic and basic groups, which are responsible for the anomalous titration behavior of native lysozyme, do not yet exist in this state. It is probable that the difference between the kinetics of refolding of lysozyme and cytochrome c may be ascribed to the existence of disulfide cross-links in lysozyme, which were intact in these experiments. These cross-links greatly limit the possible pathways for folding and thus make it much less likely that incorrectly folded states on dead-end pathways are readily accessible.

Investigations of the structure of glutamine synthetase from pig brain.

Glutamine synthetase from pig brain was purified to complete homogeneity. The sedimentation behavior, direct electron optical evidence and tracing of the difference spectra during disaggregation and denaturation by urea and detergents at different pH values and different ionic strengths lead to the conclusion that the enzyme (s= 15 S, ƒ/ƒ0= 1.26, Mr= 370000) is a dihedral, cubic octamer which consists of two enzymatically inactive half molecules (s= 8.6 S, ƒ/ƒ0= 1.36, Mr= 185000). These tetrads are formed in 2 M urea and, by dialysis against buffer, reaggregate to the native enzyme. In the tetramer, the monomers (s= 2.8 S, Mr= 46000) are in one plane, narrowly and relatively strongly associated. The dissociation of the binding forces, which is irreversible, is only possible at high concentrations of denaturants. With low concentrations of dodecylsulfate, the enzyme forms a catalytically active complex which, below pH 6.5, stacks together and precipitates in the form of helical aggregates. On addition of more dodecylsulfate, inactive dissociates arise which bind a maximum of 130 molecules of detergent per subunit. Although pig brain and sheep brain glutamine synthetases are protein‐chemically, biophysically and immunologically strikingly similar, hybridization of their tetrameric half molecules did not succeed.