Guanidine Hydrochloride Solutions Research Articles

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme.

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.

Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2×10 −4 to 6×10 −6 M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4–5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c′, cyt c 556, and cyt c 2 genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c′ and cyt c 556 have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b 562, in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe II and Fe III states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe II-cyt c 556 is more stable than the corresponding state of Fe III-cyt c 556 (ΔΔ G f°=22 kJ/mol).

Hydration at the surface of the protein Monellin: dynamics with femtosecond resolution.

We have studied the femtosecond hydration dynamics of Monellin, a protein with a single tryptophan residue at its surface. Tryptophan was selectively used as a probe of the dynamics, and through monitoring of its fluorescence Stokes shift with time we obtained the hydration correlation function, which decays due to rotational and translational motions of water at the protein surface and in bulk. The decay exhibits a "bimodal" behavior with time constants of 1.3 and 16 ps, mirroring relaxation of the free/quasifree water molecules and surface-bound water layer (minimum binding energy of 1-2 kcal/mol). The observed slow decay of 16 ps for tryptophan in the native protein differs by more than an order of magnitude from that of bulk water because of the dynamical exchange in the layer. To examine the effect of unfolding, we also studied hydration dynamics when Monellin was denatured in a 6 M guanidine hydrochloride solution and obtained a totally different behavior: 3.5 and 56 ps. Comparing with the results of experiments on free tryptophan in the same concentration of the denaturing solution, we conclude that the fast component of 3.5 ps comes from bulk-type solvation in the 6 M guanidine hydrochloride. However, the absence of the 16-ps decay and appearance of the 56-ps component reflects a more "rigid solvation," which is likely to involve the motions of the protein backbone in the random-coiled state. With the help of polymer theory, this time scale is reproduced in agreement with experimental observations.

Adsorption of Individual Polyacrylamide Molecules Studied by Atomic Force Microscopy

Adsorption of Individual Polyacrylamide Molecules Studied by Atomic Force Microscopy

Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions

It is well known that Mg 2+ is an essential component in many biological processes. This research investigated the courses of both the reactivation and the refolding in the absence and presence of Mg 2+ ions. Calf intestinal alkaline phosphatase (CIP) was extensively denatured in 3 M guanidine hydrochloride (GdnHCl) solution for 2 h. Under suitable renaturation conditions, about 60–70% of the activity was recovered in the absence and presence of different magnesium ion concentrations. The refolding processes followed two-phase courses, whereas the reactivation processes were monophasic after dilution in proper solutions with or without Mg 2+. The magnesium ions affected both the reactivation and the refolding courses of unfolded CIP. A comparison of rate constants for the refolding of unfolded CIP with those for recovery of enzyme activity at different Mg 2+ concentrations showed that they were not synchronized. The activity recovery was speeded up due to the presence of Mg 2+ ions; while the refolding course of unfolded CIP was somewhat inhibited by the excess Mg 2+.

Size and temperature dependence of hydrocarbon solubility in concentrated aqueous solutions of urea and guanidine hydrochloride

At 25°C, methane and ethane are more soluble in water than in 7 M aqueous urea or 4.9 M aqueous guanidine hydrochloride (GuHCl); the reverse is true for larger hydrocarbons. In addition, the hydrocarbon solubility in 7 M aqueous urea or 4.9 M aqueous GuHCl increases compared with that in water on raising the temperature in the range of 5–45°C. These experimental data have not yet been rationalized. Using a well-founded theory of hydrophobic hydration, the present analysis indicates that the transfer of hydrocarbons from water to 7 M aqueous urea or to 4.9 M aqueous GuHCl is favored by the difference in the solute–solvent van der Waals interaction energy, and contrasted by the difference in the work of cavity creation. At room temperature, on increasing the hydrocarbon size, the first contribution rises in magnitude more rapidly than the second contribution, accounting for the threshold size occurrence. Moreover, the second contribution decreases in magnitude with an increase in temperature, becoming less unfavorable, while the first contribution is practically constant in the range of 5–45°C. The different temperature dependence of the work of cavity creation in such solvent systems is due to the fact that the density of 7 M aqueous urea and 4.9 M aqueous GuHCl decreases more rapidly than that of water when raising the temperature. The relationship between the density of a liquid and the work to create a cavity in it is discussed in detail.Key words: work of cavity creation, solute-solvent van der Waals interaction energy, H-bond reorganization.

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus.

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.

Partial characterization of collagen in mantle and adductor of pearl oyster ( Pinctada fucata)

Histochemical observation clarified the presence of collagen in the connective tissues of epimysium, perimysium, and endomysium in the mantle and adductor of the pearl oyster Pinctada fucata. A small amount of soluble collagen could be obtained from the mantle and adductor by extracting a crude connective tissue fraction with 4 M guanidine hydrochloride (G/HCl) solution, without protease digestion of telopeptides. The G/HCl-soluble collagen showed two alpha bands (α1 and α2) on SDS–PAGE. The relative staining intensities of the α1 to α2 chains were decreased gradually by pepsin digestion with concomitant development of lower molecular weight components. These results suggest the existence of pepsin-sensitive regions in the triple helical domain of the α1 chain.

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus.

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.

A Multivalent Assay to Detect Glycosaminoglycan, Protein, Collagen, RNA, and DNA Content in Milligram Samples of Cartilage or Hydrogel-Based Repair Cartilage

The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract.

Evidence for the existence of an unfolding intermediate of thyroglobulin during denaturation by guanidine hydrochloride.

The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.

Microcalorimetric studies on the unfolding of creatine kinase induced by guanidine hydrochloride

The unfolding of rabbit muscle-type creatine kinase (MM-CK) induced by guanidine hydrochloride (GuHCl) has been studied by isothermal microcalorimetry. It has been found that the decrease in the activity of MM-CK in dilute GuHCl solution is due to a slight perturbation of the active site conformation by dilute GuHCl, but not by a reversible inhibition by GuHCl binding at the active site or dissociation of the dimeric protein. The inactivation of MM-CK precedes the overall conformation change of this enzyme during denaturation by GuHCl, providing a thermodynamic evidence for the proposition that the active site of an enzyme is situated in a limited region more flexible than the enzyme molecule as a whole. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of an intermediate state of MM-CK in the presence of moderate GuHCl concentrations at 25.00°C have been determined to be 260, 12.2 kJ mol −1, and 830 J mol −1 K −1, respectively. Further unfolding of MM-CK is observed when GuHCl concentration is higher than 3.00 mol dm −3, and the protein is almost fully unfolded at 5.00 mol dm −3 GuHCl reached. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of the unfolded state of MM-CK at 25.00°C have been measured as 8600, 23.0 kJ mol −1, and 29 kJ mol −1 K −1, respectively. The experimental results indicate that the unfolding of MM-CK by GuHCl exhibits remarkable enthalpy–entropy compensation and the water reorganization is involved in the unfolding reaction.

Non-polar solutes in water and in aqueous solutions of protein denaturants: modeling of solution and transfer processes

A simple molecular model for the thermodynamic behavior of non-polar solutes in water and in aqueous solutions of protein denaturants is presented. Three contributions are considered: (i) combinatorial arising from the mixing process, (ii) interactional characterizing the molecular interactions occurring in the mixture and (iii) a contribution originating from the structural changes occurring in the first shell of water molecules around the solute. The latter is modeled assuming that water molecules in contact with the solute are involved in a chemical equilibrium between two states. The model describes well the temperature and denaturant concentration dependences of the Gibbs energies of solution and transfer for benzene, toluene and alkanes in water and aqueous solutions of urea and guanidine hydrochloride. Model parameters are physically meaningful, allowing a discussion of the molecular interactions involved. A preferential solvation of the solute by the denaturant is found. However, the non-polar solute-denaturant interaction is not specific, i.e. leading to a distinct chemical entity. Urea and guanidine hydrochloride are non-polar solubilizing agents because their interactions with the solute are less unfavorable than those between water and the solute.

Inhibition of Alzheimer's disease beta-amyloid aggregation, neurotoxicity, and in vivo deposition by nitrophenols: implications for Alzheimer's therapy.

SPECIFIC AIMSWe have examined the hypothesis that small molecular weight compounds capable of inhibiting the aggregation of the beta-amyloid peptide (Aβ) could protect neurons from the toxic effect of Aβ and prevent cerebral amyloid deposition. The anti-aggregating and neuroprotective effects of nitrophenols were investigated in vitro using primary cultures of rat hippocampal neurons, and the capacity to inhibit amyloid formation was evaluated in vivo in rats using a model system of cerebral amyloid deposition.PRINCIPAL FINDINGS1. Aβ fibrils are stabilized by hydrophobic interactions mediated by the carboxyl-terminal domain of the peptideAmyloid fibrils were formed in vitro by dissolving either full-length Aβ (Aβ1-42 and Aβ1-43) or the carboxyl-terminally truncated Aβ1–28 peptide in aqueous buffer, and their stabilities in denaturant solutions were investigated. We found that the stability of fibrillar Aβ in guanidine hydrochloride (GdnHCl) solutions was markedly dependent on peptide chain length. For Aβ1...

Barnacle cement proteins. Importance of disulfide bonds in their insolubility.

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.

The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding.

Viscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228-3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co-solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.

Change in backbone torsion angle distribution on protein folding

Understanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic-detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 A(-1). Denaturation leads to a reduction in the protein atom-pair distance distribution function over the approximately 3-15 A region that is associated with a quantifiable shift in the backbone torsional angle (phi, psi) distribution toward the beta region of the Ramachandran plot.

Adhesive properties of soy proteins modified by urea and guanidine hydrochloride

AbstractAn investigation was conducted on the adhesive and water‐resistance properties of soy protein isolates that were modified by varying solutions of urea (1, 3, 5, and 8 M) or guanidine hydrochloride (GH) (0.5, 1, and 3 M) and applied on walnut, cherry, and pine plywoods. Soy proteins modified by 1 and 3 M urea showed greater shear strengths than did unmodified protein. The 3 M urea modification gave soy protein the highest shear strength. Soy proteins modified with 0.5 and 1 M GH gave greater shear strengths than did the unmodified protein. The 1 M GH‐modified soy protein gave the highest shear strength. Compared to the unmodified protein, the modified proteins also exhibited higher shear strengths after incubating with two cycles of alternating relative humidity, zero delamination, and higher remaining shear strengths after three cycles water soaking and drying. These results indicate that soy proteins modified with urea and GH enhance water resistance as well as adhesive strength. Secondary structures of globule proteins may enhance adhesion strength, and the exposure of hydrophobic amino acids may enhance water resistance. Proteins modified by 3 M urea or 1 M GH may have higher content of secondary structure and more exposed hydrophobic amino acids, compared with other modifications or unmodified proteins.

The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ((2)H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C(1) or C(5) of the beta-ionone ring or carbon C(9) of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. (2)H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 degrees C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 degrees C and then rapidly cooled to -50 degrees C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the (2)H NMR line shapes, the angles between the individual C-CD(3) bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C(5)-C(13) part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C(9)-CD(3) bond shows the largest orientational change of 7 degrees in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C(9)-methyl group and Trp 182 in helix F.