The solubilization of progesterone by mixed bile,salt-phospholipid sols

The solubility of progesterone was determined in several different bile salt-phospholipid mixtures, and it is concluded that: (1) The solubility in unconjugated bile salts is greater than in the conjugated analogues, and the solubility in deoxycholate solutions is twice that in cholate solutions. (2) Substitution of hydroxyl groups in the 11 and 21 positions of progesterone increases solubility, whilst substitution in the 17-position decreases solubility in bile salt solutions. (3) Progesterone solubility in mixed bile salt solutions is proportional to the mole ratio of the surfactant mixture. (4) Sodium deoxycholate (SDC)-phospholipid sols show no such linear solubilizing properties; a minimum occurring at a mole ratio of SDC to phospholipid of 1 : 4. (5) There is a break in the solubility curve of progesterone in lysophosphatidycholine (LPC)/phosphatidylcholine (PC) mixtures at a mole ratio of 65 : 35 coincident with maximum viscosity. (6) Introduction of SDC into LPC/PC mixtures results in decreased progesterone solubility.     

Partial specific volumes and interactions with solvent components of α-globulin fromSesamum indicum L. in urea and guanidine hydrochloride

The interaction of α-globulin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volumes of the protein in these solvents. The apparent partial specific volumes were determined both under isomolal and isopotential conditions. The preferential interaction parameter with solvent components calculated were 0.08 and 0.1 g of urea and guanidine hydrochloride respectively per g protein. In both the cases the interaction was not preferential with water. The total binding of denaturant to α-globulin was calculated both for urea and guanidine hydrochloride and the correlation between experimentally determined number of mol of denaturant bound per mol of protein and the total number of peptide bonds and aromatic amino acids were found to be in excellent agreement with each other. The changes in volume upon transferring α-globulin from a salt solution to 8 M urea and 6 M guanidine hydrochloride were also calculated. This work was done at the Biochemistry Department, Brandeis University, Waltham, Massachusetts 02254, USA.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.     

Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

By analyzing the effect of urea and guanidine hydrochloride on the circular dichroism of many polypeptides and proteins, it is concluded that under conditions of high concentration of the perturbant and at low temperatures the resultant state approached is that of a local extended helix structure instead of a completely random coil. Intensification by urea and guanidine hydrochloride of the circular dichroism bands of poly-L -proline II leads to the proof that the mechanism of interaction of urea and guanidine hydrochloride with proteins is through hydrogen bonding to the backbone carbonyl group.     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions.

Previous work has shown that at neutral pH ferricytochrome c (horse heart) retains certain residual structures in concentrated solutions of urea or guanidine hydrochloride (Tsong, T. Y. (1974), J. Biol. Chem. 249, 1988). Present studies reveal that cooperative unfolding of these residual structures can be achieved by acidification of the protein to pH 4 in 9 M urea but can only be partially achieved in a 6 M guanidine hydrochloride solution. The evidence that the residual structures unfold in 9 M urea upon acidification is twofold. (1) Further uncoupling of the Trp-59-heme interaction occurs; this is reflected in the intensification of the tryptophan fluorescence from 55 to 90 percent relative to that of free tryptophan in the same solvent. (2) The intrinsic viscosity of the protein solution increases from 15.0 to 21 ml/g. The acidification also induces a spin-state transformation of the heme group at pH 5 both in urea and in guanidine hydrochloride. Acidic titration of the protein in urea and guanidine hydrochloride indicates that the unfolding involves the absorption of a single proton. However, the kinetics of the spin-state transformation are triphasic. These results suggest that the displacement of the ligand His-18 by a solvent molecule and the subsequent disintegration of the residual structures are complex processes and involve at least three kinetic steps. The ineffectiveness of guanidine hydrochloride as a denaturant for ferricytochrome c is shown to be due to the presence of the high concentration of Cl minus which can stabilize certain elements of the protein structure.     

Rapid and efficient extraction of soluble proteins from gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably permeate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

Rapid and efficient extraction of soluble proteins from Gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably penetrate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Interactions of biocidal guanidine hydrochloride polymer analogs with model membranes: a comparative biophysical study

Four synthesized biocidal guanidine hydrochloride polymers with different alkyl chain length, including polyhexamethylene guanidine hydrochloride and its three new analogs, were used to investigate their interactions with phospholipids vesicles mimicking bacterial membrane. Characterization was conducted by using fluorescence dye leakage, isothermal titration calorimetry, and differential scanning calorimetry. The results showed that the gradually lengthened alkyl chain of the polymer increased the biocidal activity, accompanied with the increased dye leakage rate and the increased binding constant and energy change value of polymer-membrane interaction. The polymer-membrane interaction induced the change of pretransition and main phase transition (decreased temperature and increased width) of phospholipids vesicles, suggesting the conformational change in the phospholipids headgroups and disordering in the hydrophobic regions of lipid membranes. The above information revealed that the membrane disruption actions of guanidine hydrochloride polymers are the results of the polymer's strong binding to the phospholipids membrane and the subsequent perturbations of the polar headgroups and hydrophobic core region of the phospholipids membrane. The alkyl chain structure significantly affects the binding constant and energy change value of the polymer-membrane interactions and the perturbation extent of the phospholipids membrane, which lead to the different biocidal activity of the polymer analogs. This work provides important information about the membrane disruption action mechanism of biocidal guanidine hydrochloride polymers.     

Two structurally different types of rabbit light chains

Disulphide bonds of rabbit γ-G-globulin and the antibody of the γ-G-globulin type against the 2,4-dinitrophenyl group were split both by the oxidative sulphitolysis at pH 8.6 and by the reduction with 2-mercaptoethanol followed by carboxymethylation. The fractionation was carried out in 0.05 m formic acid containing 6m urea, in 1m propionic acid or in 6m guanidine hydrochloride. Both heavy (H) and light) (L) chains are released from the I+J fraction preceding on an elution diagram H chains when rechromatographed in a stronger desaggregation medium. A small amount of the L chains is also released on rechromatography of the H chains (isolated from 1m propionic acid) in 6m guanidine hydrochloride. The separation of the degraded γ-G-globulin in 0.05m formic acid containing 6m urea or in 6m guanidine hydrochloride showed a separation of the L chains to two fractions differing by electrophoretic properties, peptide maps and N-terminal amino acids. However, these chains exhibit a similar molecular weight, immunoelectrophoretic behaviour and similar properties on reactivation of the antibody H chain.     

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.     

Equilibrium unfolding and conformational plasticity of troponin I and T.

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis-ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis-ANS, indicating the existence of solvent-exposed hydrophobic domains in their structures. Bis-ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far-UV CD and bis-ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states. Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.     

Two-domain structure of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase.

NADH-cytochrome b₅ reductase is an amphiphilic protein consisting of a hydrophilic (FAD-containing) moiety and a hydrophobic (membrane-binding) segment and exists in aqueous media as an oligomeric aggregate. Circular dichroism studies have shown that denaturation of the reductase by guanidine hydrochloride in the presence of Emulgen 109P, a nonionic detergent, is a two-stage process as a function of the denaturant concentration. The first transition occurs at about 1 m guanidine hydrochloride and the second one at much higher concentrations. The guanidine hydrochloride concentration causing the second-stage unfolding depends on the concentration of Emulgen 109P. A hydrophilic fragment of the reductase lacking the hydrophobic segment undergoes one-stage denaturation at about 1 m guandine hydrochloride regardless of the presence and absence of Emulgen 109P. Both the reductase as well as the hydrophilic fragment lose their NADH-ferricyanide reductase activity and FAD also at about 1 m guanidine hydrochloride in the presence of the detergent. These findings suggest that the first-stage denaturation of the reductase represents the unfolding of the hydrophilic moiety and the second one that of the hydrophobic segment. Gel chromatography experiments have suggested that in the presence of Emulgen 109P the reductase exists as a mixed micelle with the detergent and this aggregation state persists even after the first-stage denaturation (unfolding of the hydrophilic moiety). The dissociation of the mixed micelle seems to take place concomitant with the second-stage denaturation. It is concluded that the two moieties of the reductase molecule, though linked to each other covalently, exist as independent domains undergoing unfolding separately at least in the presence of Emulgen 109P. This structural feature of the reductase is similar to that of cytochrome b₅ reported by us. The reductase is, therefore, another example of amphiphilic membrane proteins having two structurally independent domains in the molecule.     

Competing solvent effects of polyols and guanidine hydrochloride on protein stability

The denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25 degrees C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. These results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.     

Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas

Thiol proteinase inhibitors are crucial to proper functioning of all living tissues consequent to their cathepsin regulatory and myriad important biologic properties. Equilibrium denaturation of dimeric goat pancreas thiol proteinase inhibitor (PTPI), a cystatin superfamily variant has been studied by monitoring changes in the protein's spectroscopic and functional characteristics. Denaturation of PTPI in guanidine hydrochloride and urea resulted in altered intrinsic fluorescence emission spectrum, diminished negative circular dichroism, and loss of its papain inhibitory potential. Native like spectroscopic properties and inhibitory activity are only partially restored when denaturant is diluted from guanidine hydrochloride unfolded samples demonstrating that process is partially reversible. Coincidence of transition curves and dependence of transition midpoint (3.2M) on protein concentration in guanidine hydrochloride‐induced denaturation are consistent with a two‐state model involving a native like dimer and denatured monomer. On the contrary, urea‐induced unfolding of PTPI is a multiphasic process with indiscernible intermediates. The studies demonstrate that functional conformation and stability are governed by both ionic and hydrophobic interactions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 708–717, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.