GuHCl Concentration Research Articles

Inactivation before significant conformational change during denaturation of papain by guanidine hydrochloride

During denaturation by GuHCl, papain shows a rapid decrease in activity with increasing concentrations of the denaturant followed by an intermediate stage of relatively little change from 1 to 2 M before complete inactivation at 4 M GuHCl. At GuHCl concentrations lower than 2 M, enzyme activity is more sensitive to GuHCl than noticeable conformation changes as followed by fluorescence and CD measurements. Kinetics of GuHCl inactivation were studied by following the substrate reaction in the presence of denaturant and the apparent rate constants thus obtained were found to be only slightly higher than those for conformational changes. However, apparent inactivation rate constants obtained in the presence of saturating concentration of substrate are actually inactivation constants for the ES complex. The inactivation rates at different substrate concentrations were, therefore, followed and the microscopic inactivation rate constants for the free enzyme obtained (Tsou, C.L. (1988) Adv. Enzymol. 61, 381–436).It was found that substrate protects strongly against inactivation and at the same GuHCl concentration, the inactivation rate of the free enzyme is 100-fold higher than that of unfolding. The above results show that the activity of papain is more sensitive to GuHCl than its overall conformation and like the enzymes previously studied in this laboratory, its active site is more flexible than the enzyme molecule as a whole.

Characterization of folding intermediates of human carbonic anhydrase II: probing substructure by chemical labeling of SH groups introduced by site-directed mutagenesis.

By measurement of UV absorbance, CD spectra, and enzyme activity, we have shown that human carbonic anhydrase II forms a stable and compact folding intermediate at a moderate concentration of guanidine hydrochloride. The major aim of this study was to map the intermediate structure. For that reason, site-directed mutagenesis was used to introduce cysteine residues in various parts of the central beta-structure to give in each case a single cysteine residue. Thereafter, the accessibility of the introduced SH group to specific chemical labeling was used to probe the stability and compactness of the area surrounding each cysteine residue. Our results indicate that the folding intermediate has an ordered native-like secondary structure in the central part of the beta-sheet, whereas the peripheral part of the beta-sheet seems to be less ordered. A large hydrophobic cluster situated between the central beta-sheet core and secondary structure elements on the surface appears to be intact in the intermediate and is remarkably stable even at high GuHCl concentrations (> 5 M). This unusually stable substructure might function as a "seed" during the initiation of the folding process.

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.

Compact denatured state of a staphylococcal nuclease mutant by guanidinium as determined by resonance energy transfer.

The protein from a mutant clone of staphylococcal nuclease with a cysteine substituting for a lysine at position 78 was prepared and labeled with a cysteine-specific fluorescent probe 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). Time-resolved nonradiative energy-transfer studies were done using the single tryptophan at position 140 as the energy donor and the IAEDANS as the receptor. Changes in distance and distance distributions were observed as a function of increasing guanidinium (GuHCl) concentration (0-2 M) and in the presence or absence of Ca2+ and inhibitor 2'-deoxythymidine 3',5'-diphosphate (pdTp). In the native state, both the ternary complex and the noncomplexed protein are best fit with one population having an average donor-acceptor distance of approximately 23 A and an "apparent" full width at half-maximum (fwhm) of distance distribution of approximately 18 A. Besides the contribution of linker arm of the acceptor, it appears that there are some conformational heterogeneties either due to the disordering of the tryptophan region or due to the whole protein in the native state. During GuHCl unfolding, the average distance remains relatively constant up to GuHCl concentrations where both the ternary complex and the ligand-free protein are denatured (1-1.3 M). The compact denatured states persist up to 2 M GuHCl. At 2 M GuHCl, the heterogeneity of the denatured state in the ternary complex is much larger than that of the ligand-free nuclease. The results show that the denatured states of staphylococcal nuclease mutant K78C by GuHCl are compact and these compact denatured states are likely due to residual structures or incompletely disrupted hydrophobic cores under these conditions.

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.

Effect of porcine bone morphogenetic protein on healing of bone defect in the rabbit radius.

Segmental long bone defects due to infection or trauma is a difficult problem to manage in patients. We studied the effect of porcine bone morphogenetic protein (pBMP) on healing of defects in the rabbit radius. Porcine BMP was separated and purified from the tibia and femur of pigs by repeated solubilization and precipitation of the protein with different concentrations of urea and GuHCl. The osteoinductive activity of pBMP was confirmed by bioassay using No. 615 mice. In rabbits, about a 15 mm length of radii were removed and 20 mg of pBMP was implanted in the defected area with fibrin sealant (FS), while only FS was implanted in controls. Union of the affected area was observed in 6 weeks in the experimental side. There was no definite evidence of bone bridging across the affected area in the controls. This suggests that pBMP has a bone forming activity in other species and the clinical use of pBMP in treating patients with segmental bone defects is promising.

Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.

GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.

Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

Studies on the Extraction of Different Proteoglycan Populations in Bovine Articular Cartilage

Large proteoglycan monomers and small dermatan sulfate proteoglycans were extracted from explants of bovine articular cartilage with increasing (0-4 M) concentrations of guanidinium chloride (GuHCl). The first extractions were followed by a second extraction with 4 M GuHCl. The amount of proteoglycans extracted in the first buffer depended on the GuHCl concentration. At low concentrations of GuHCl, a relatively high amount of small proteoglycans was obtained. Fifty percent of the small proteoglycans was extracted in buffer with 0.85 M GuHCl, while 2.0-2.2 M GuHCl was needed to extract half of the large proteoglycans. Immediately after synthesis, 35S-labeled large proteoglycans were extracted much easier (50% at 1.4 M GuHCl), and those extracted at low concentrations of GuHCl were less capable of aggregation with hyaluronic acid. After 7 days of 'chase' these differences between endogenous and 35S-labeled proteoglycans had disappeared.

Separation and Purification of Porcine Bone Morphogenetic Protein

Porcine bone morphogenetic protein (pBMP) was separated and purified from porcine bone by repeated solubilization and precipitation of the protein with different concentrations of urea and GuHCl. The molecular weight of pBMP was determined to be 19.7 k. The property of inductive osteogenesis of pBMP caused new bone formation in muscular and subcutaneous tissues of strain No. 615 mice. Osteoid tissues were observed as early as ten days after implantation of pBMP, and lamellated new bones with marrow cavities were first found on the 15th day of implantation. A gradual transformation of mesenchymal-type cells into active chondroblasts and osteoblasts and finally into heterotopic new bone was observed and studied histologically and ultrastructurally.

Association-dissociation of histone oligomers. A spin label study.

The spin label method has been used to obtain information about conformational changes of histone oligomers taking advantage of the fact that at a low ionic strength and in the presence of other histones about 45% of cysteine residues of histone H3 react with the 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl spin label. For the labeled complexes H3-H4 and H nu the degree of immobilization of the spin label is a function of the ionic strength. This variation is identical for both complexes within a long range of ionic strengths, including the interval of 0.8-2 M NaCl, under which conditions interactions are known to exist between the tetramer (H3)2 (H4)2 and the dimer (H2A) (H2B). This finding suggests a negligible influence of the dimer for modifying the cysteine residue environment of histone H3 on octamer formation. GuHCl treatment at high ionic strength of the labeled complexes gives rise to a non-lineal increase in the degree of mobility of the spin label. This increase, at low GuHCl concentration (0-0.5 M GuHCl), is interpreted as showing a lowering in rigidity for the Cys residue environment, without affecting the general stability of the tetramer (H3)2 (H4)2. At higher GuHCl concentration (2-3 M GuHCl) the increase in the spin label mobility is related to a dissociation of the complexes in single histones. Our results are consistent with the view that the overall structure of the tetramer, as well as its conformational changes during complex structuration or denaturation, are not strongly affected by the presence of the dimer (H2A) (H2B).

Characterization of the transition state of lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state.

AbstractThe influence of proline cis‐trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two‐state transition model well. The temperature dependencies of kuf and kf over a wide temperature range showed that ΔC = 0 and ΔC = −6.7 kJ K−1 mol−1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol−1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol−1 is primarily required for the disruption of long‐range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.

α-lactalbumin: compact state with fluctuating tertiary structure?

Gu-HCl shows a non-simultaneous change of different optical properties: the CD spectrum in near UV (aromatic) region changes at smaller concentrations of Gu-HCl than the CD spectrum in far UV (peptide) region [l--4]. This suggests the existence of stable, partly denatured (intermediate) forms of these proteins with a more or less symmetrical environment of aromatic groups but with a more or less native-like secondary structure. For both bovine [2] and human [4] a-lactalbumins as well as for some other proteins [S] a similar state exists also at acid pH. While the partly denatured (P) form at moderate Gu-HCl concentrations usually can be obtained only in mixture with native (N) and/or unfolded (U) forms, the acid (A) forms of these proteins can be studied in pure state. It was shown that for bovine (u-LA U + A transition takes <l ms while A + N transition needs as much as 0.1-I s which suggests that the intermediate state similar to the A-form can be also a kinetic intermediate in the course of protein folding [6]. nounced than that of the native protein. This paper presents the results of investigation of acid and some similar forms of bovine and human a-lactalbumins which show that these forms are compact, have secondary structure similar to the native one but their tertiary structure can slowly fluctuate. More detailed experimental data and their discussion will be published later.

Studies on groundnut proteins V. Physico-chemical properties of arachin prepared by different methods

ORD, 1 1 Abbreviations used: ORD, optical rotatory dispersion; CD, circular dichroism; SDS, sodium dodecyl sulphate; GuHCl, guanidinium hydrochloride; EDTA, ethylene diamine tetracetic acid. CD, and fluorescence spectra of arachin prepared by Tombs' ( Biochem. J., 96, 119; 1965), Dawson's ( Anal. Biochem., 41, 305; 1971) or Shetty and Rao's ( Anal. Biochem., 62, 108; 1974) procedure were measured; the effect of denaturants such as SDS, GuHCl, and acid was also determined. ORD and CD spectra showed differences, whereas fluorescence spectra did not show any difference. The effect of the denaturants was the same on the three arachins. At low concentrations of GuHCl (<2 m), the denaturant was bound by the protein molecule without causing any conformational change. The binding affinity varied among the arachins.

Selective histochemical staining of cartilage matrix by Rivanol.

AbstractRivanolR, a fluorescent ethoxy derivative of acridine, interacts at different pH's with both glycosaminoglycans and proteins. The present study utilizes the specific interaction of RivanolR with acidic substances of the ground substance for histochemical studies of the cartilage matrix. This stain was applied to newborn mouse epiphyseal cartilages which were either unextracted or dissociatively extracted by graded concentrations of guanidinium chloride (GuHCl) from 0.5–3.0 M for four days at 25°C. Routinely prepared sections were then stained (0.1% solution) for two minutes at pH's ranging from 2.2–11.2. Stainability of the interterritorial matrix as well as the inner halo zone and outer corona zone of the lacunar matrix varied with pH. Whereas the interterritorial matrix decreased in stainability with rising pH, the halo and corona persisted in stainability up to pH 10.7. Dissociative extractions using GuHCl revealed the unextractable nature of the inner halo zone as well as the extractable nature of the corona above 1.0 M GuHCl concentration. Anionic sites on polyelectrolytes such as glycosaminoglycans are known to stoichiometrically bind many cationic dyes. The precise localization of stain‐reactive glycosaminoglycans or proteoglycans in the region of the perichondrocytic matrix by RivanolR supports prior observations using other cationic stains. Our data demonstrates that RivanolR enables one to visualize the unique perichondrocytic matrix which may be interpreted to be both chemically and morphologically a “matrix within a matrix”.