Effects Of Guanidine Hydrochloride Research Articles

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Structure and stability of D-galactose/D-glucose-binding protein. The role of D-glucose binding and Ca ion depletion

The effects of guanidine hydrochloride (GdnHCl) on the structure and stability of the D-galactose/D-glucose-binding protein from Escherichia coli (GGBP) and its complex with D-glucose (GGBP/Glc) were investigated by intrinsic protein fluorescence and far-UV circular dichroism (CD). The role of calcium in the stability of the protein structure was also studied. It was shown that the processes of GGBP and GGBP/Glc unfolding induced by GdnHCl followed one-step reversible denaturation mechanism. The obtained data showed that the binding of glucose to GGBP resulted in an increase of the protein stability towards the actions of the GdnHCl which made protein unfolding more cooperative. The stabilities of GGBP alone, GGBP in the presence of glucose, GGBP-depleted calcium (GGBP-Ca), and GGBP/Glc-depleted calcium (GGBP/Glc-Ca) were characterized by difference of Gibbs free energies.

Structure and stability of D-galactose/D-glucose-binding protein. The role of D-glucose binding and Ca ion depletion

The effects of guanidine hydrochloride (GdnHCl) on the structure and stability of the D-galactose/D-glucose-binding protein fromEscherichia coli(GGBP) and its complex with D-glucose (GGBP/Glc) were investigated by intrinsic protein fluorescence and far-UV circular dichroism (CD). The role of calcium in the stability of the protein structure was also studied. It was shown that the processes of GGBP and GGBP/Glc unfolding induced by GdnHCl followed one-step reversible denaturation mechanism. The obtained data showed that the binding of glucose to GGBP resulted in an increase of the protein stability towards the actions of the GdnHCl which made protein unfolding more cooperative. The stabilities of GGBP alone, GGBP in the presence of glucose, GGBP-depleted calcium (GGBP-Ca), and GGBP/Glc-depleted calcium (GGBP/Glc-Ca) were characterized by difference of Gibbs free energies.

Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy

Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of lambda(max) point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (approximately 5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment.

Effects of Guanidine Hydrochloride on Pyruvate Dehydrogenase Complex

Effects of Guanidine Hydrochloride on Pyruvate Dehydrogenase Complex

Effects of guanidine hydrochloride on the proton inventory of proteins: implications on interpretations of protein stability.

The DeltaG degrees (N)(-)(D) value obtained from extrapolation to zero denaturant concentration by the linear extrapolation method (LEM) is commonly interpreted to represent the Gibbs energy difference between native (N) and denatured (D) ensembles at the limit of zero denaturant concentration. For DeltaG degrees (N)(-)(D) to be interpreted solely in terms of N and D, as is common practice, it must be shown to be independent of denaturant concentration. Because DeltaG degrees (N)(-)(D) is often observed to be dependent on the nature of the denaturant, it is necessary to determine the circumstances under which DeltaG degrees (N)(-)(D) can be interpreted as a property solely of the protein. Here, we use proton inventory, a thermodynamic property of both the native and denatured ensembles, to monitor the thermodynamic character of denaturant-dependent aspects of N and D ensembles and the N right arrow over left arrow D transition. Use of a thermodynamic rather than a spectral parameter to monitor denaturation provides insight into the manner in which denaturant affects the meaning of DeltaG degrees (N)(-)(D) and the nature of the N right arrow over left arrow D transition. Three classes of proteins are defined in terms of the thermodynamic behaviors of their N right arrow over left arrow D transition and N and D ensembles. With guanidine hydrochloride as a denaturant, the classification of protein denaturations by these procedures determines when the LEM gives readily interpretable DeltaG degrees (N)(-)(D) values with this denaturant and when it does not.

NMR studies on dynamic behavior of water molecule in aqueous denaturant solutions at 25 °C: Effects of guanidine hydrochloride, urea and alkylated ureas

Abstract The spin-lattice relaxation times (T1) of the 17O nucleus of a water molecule in guanidine hydrochloride, urea, and alkylated ureas at 25 °C were measured by NMR spectroscopy. Urea has no significant effect on the water structure. Also, guanidine hydrochloride as a stronger denaturant than urea does not perturb the water structure similar to urea. The change in the dynamic structure (BR) of water was proportional to the carbon number of the alkyl chain of the alkylated urea. Compared to the results of alcohols and tetraalkylammonium solution, it is clear that the contribution of one carbon of the alkyl chain to the water structure is independent of the type of solute. The BR of urea and its alkylated substances show a good correlation with the concentration of denaturation midpoints obtained on sperm whale myoglobin and cytochrome c. Because the water structure is not perturbed by urea and guanidine hydrochloride, these results indicate that the effect of hydrophobic hydration of denaturants becomes important for the denaturation of a protein with increasing hydrophobicity of the denaturants.

The effects of guanidine hydrochloride on the 'random coil' conformations and NMR chemical shifts of the peptide series GGXGG.

The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH(2). The phi angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis-trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of(3)J(HNHalpha) values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of 'random coil' (1)H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that (13)C and(15)N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.

Independent folding of the domains in the hydrophilic subunit IIABman of the mannose transporter of Escherichia coli.

The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli is a homodimer of two 35-kDa subunits. Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion. Separation of tryptic fragments yields monomers of IIB and dimers of IIA, which are active and stable. To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments. Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each isolated fragment. The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and second transitions of intact IIABman. Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA. Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments. The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions. The lack of interdomain interactions suggests an unrestricted domain motion. This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain).

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins.

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.

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.

Characterization of the critical state in protein folding: Effects of guanidine hydrochloride and specific Ca 2+ binding on the folding kinetics of α-lactalbumin

The reversible unfolding and refolding kinetics of α-lactalbumin induced by concentration jump of guanidine hydrochloride were measured at pH 7.0 and 25 °C using tryptophan absorption at 292 nm, with varying concentrations of the denaturant and free Ca 2+. The refolding reaction of α-lactalbumin from the fully unfolded (D) state occurs through the two stages: (1) instantaneous formation of a compact intermediate (the A state) that has a native-like secondary structure; (2) tight packing of the preformed secondary structure segments to lead finally to the native structure, this stage being the rate-determining step of the reaction and associated with aquisition of the specific structure necessary for strong Ca 2+ binding. Under strongly native conditions, the observed kinetics of refolding is also complicated by the presence of a slow-folding species (10%) in the unfolded state. Considering these facts, the microscopic rate constants in folding and unfolding directions have been evaluated from the observed kinetics and from the equilibrium constants of the transitions among the native (N), A and D states. Close linear relationships have been found in the plots of the activation free energies, obtained from the microscopic rate constants, against the denaturant concentration. They are similar to the linear relationship between the free energy of unfolding and the denaturant concentration. It was demonstrated that the slope of the plots should be approximately proportional to a change in accessible surface area of the protein during the respective activation process, and that only a third of the difference in accessible surface area between A and N is buried in the critical activated state of folding. However, the selective effect of Ca 2+ binding on the folding rate constant has been observed also, demonstrating that the specific Ca 2+-binding substructure in the N state is already organized in the activated state. Thus, only a part of the protein molecule involving the Ca 2+-binding region is organized in the activated state, with the other part of the molecule being left less organized, suggesting that the second stage of folding may be a sequential growing process of organized assemblage of the preformed secondary structure segments.

Effects of Guanidine Hydrochloride on Electroless Copper Deposition

The effects of guanidine hydrochloride on electroless copper plating were studied by cyclic voltammetry and Tafel plot. An electrode from electroless deposited copper was used as the working electrode in these experiments. It was found that guanidine hydrochloride influences the plating process thermodynamically and kinetically. The surface composition and morphology of electroplated copper and electroless copper were studied by Auger spectroscopy and scanning electron microscope. The morphology of the electroless copper becomes rougher and porous because of guanidine hydrochloride. This is the other reason why guanidine hydrochloride enhances electroless copper deposition rate.

Second derivative spectral properties of tryptophan and tyrosine residues in proteins. Effects of guanidine hydrochloride and dodecyl sulfate on the residues in lysozyme, ribonuclease and serum albumin.

The 2nd derivative spectra of tryptophan and tyrosine, and of ribonuclease (as a tyrosine-rich protein) and lysozyme (as a tryptophan-rich protein) were recorded under various conditions. On the basis of these spectra, the spectral bands of tryptophan and tyrosine residues in proteins were analyzed. Furthermore, the effects of the anionic surfactant sodium dodecyl sulfate on the environment around tryptophan and tyrosine residues in bovine serum albumin were studied.

Expression of functionality of alpha-chymotrypsin. Effects of guanidine hydrochloride and urea in the onset of denaturation.

Crystals of alpha-chymotrypsin (CHT) at equilibrium in solutions of 2.0 M guanidine hydrochloride and 3.0 M urea at pH 3.6 were prepared, three-dimensional X-ray intensities were measured, and difference electron-density maps were calculated and examined. The guanidine hydrochloride derivative displayed changes occurring exclusively on the surface of the protein. The difference peaks represented mostly small changes in various protein surface groups and in the adjacent solvent regions, and some displayed convincing evidence of binding of the guanidinium ion to the protein. The urea difference map likewise showed that changes had occurred on the surface of the protein, but also that numerous changes in the structure occurred in the hydrophobic interior of the CHT molecule. Further, the urea difference map contained evidence for two kinds of interactions of urea with protein groups. There are examples of bound urea either causing or accompanying structural changes and examples of urea binding with no accompanying changes to the protein. Examples of both kinds of binding were observed in both the surface regions and in the hydrophobic interior of the molecule. From an examination of these two derivatives, it is clear that guanidine hydrochloride and urea unfold proteins by different mechanisms.

Structural stability of glycophorin: Effects of heat and guanidine · HCl

The effects of guanidine hydrochloride and high temperature on human glycophorin and sialic acid-free glycophorin were monitored by circular dichroism, viscosity, and fluorescence of 1-anilino-8-naphthalane sulfonate (ANS). The following observations were made: 1. 1.|Glycophorin and its sialic acid-free counterpart are unusually stable to both guanidine · HCl and heat. 2. 2.|CD and viscosity measurements indicate that guanidine · HCl neither causes a cooperative unfolding nor generates a random coil. 3. 3.|The ANS binding site is much more sensitive to guanidine · HCl than the ellipticity at 220 nm ( θ 220). 4. 4.|The effect of temperature on CD is reversible whereas the effect of guanidine · HCl is not. 5. 5.|The carbohydrate moiety influences the viscosity, and also contributes to the changes in θ 220 when solutions of glycophorin are heated. These unusual properties indicate a complex mechanism of unfolding for this structurally stable macromolecule.

Effects of guanidine hydrochloride on human plasma high density lipoproteins

Denaturation of human plasma high density lipoproteins during ultracentrifugation in guanidine-HCl is characterized by: dissociation of apoA-I, in the range of 2–3 M guanidine-HCl, and dissociation of apoA-I and apoA-II in 5–6 M guanidine-HCl. Denaturation of high density lipoprotein species, during a sequence of timed exposure to guanidine-HCl followed first by removal of the denaturant by dialysis and then by ultracentrifugation, is characterized by: dissociation of lipid-poor apoA-I, which follows a time course similar to denaturation-related changes in reported spectroscopic parameters; and apparent formation of lipoprotein aggregation products depleted in apoA-I and relatively enriched in apoA-II. These studies indicate differential properties of the major apoproteins in stabilizing high density lipoprotein structure and characterize a mode of lipoprotein transformation and degradation which apparently results from apoprotein dissociation coupled with aggregation of denatured lipoprotein species.

Controlled alterations in the physical and biological properties of R17 bacteriophage induced by guanidine hydrochloride

The effects of guanidine hydrochloride on the physical and biological properties of R17 phage were studied to ascertain how these effects relate to the structure of the phage particle. The dialysis of phage against 3.0 M guanidine at 4 C resulted in a loss of infectivity and subtle changes in the sedimentation properties of the resulting non-infectious particles. Upon increasing the temperature of the reaction from 4 C to 37 C, additional physical modifications were found to occur in a sequential manner, giving rise to three unique particle forms followed by the eventual breakdown of phage structure. These three modified phage forms were found to have S values of 78, 58, and 44, respectively, to contain all three macromolecules characteristic of R17 phage, and to have the buoyant density of normal phage. The RNA of these particles were sensitive to RNase, and negative stains readily penetrated their interior, suggesting that changes had occurred in the capsid. The 44S particles in particular were found to be highly unstable as judged by electron microscopic and analytical ultracentrifugational studies. Various stages in the disassembly of these particles were identified. Protein fragments directly released from 44S particles were found to be morphologically similar to 11S subunits produced during the breakdown of phage in higher concentrations of guanidine. The morphological identify of the subunits released from 44S particles and the 11S protein subunits was supported by the similarity of the two types of subunits in forming branched chains, in the arrangement of subunits within the chains, and by their cosedimentation in sucrose gradients. Further analysis of the 11S subunits showed the following characteristics: (1) three morphological configurations with the most common form having dimensions of 106 × 50 Å; (2) a sedimentation rate of 10.5–11.4S; (3) molecular weight of 240,000 daltons. On the basis of these properties and on the known properties of phage coat protein, a model is proposed to explain the structure of these subunits and their possible relalationship to the structure of the phage particle. Additional support for the proposed model of phage structure is provided by electron microscopic observations and the recently discovered functions of the A (maturation) protein.