High Concentrations Of Guanidine Hydrochloride Research Articles

Isotopic exchange plus substrate and inhibition kinetics of D-xylose isomerase do not support a proton-transfer mechanism.

The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.

Detection of Electrophoretic Variants of Notch, PS Integrin, and DROP-1 Proteins in Drosophila melanogaster Following Extraction in Guanidine Hydrochloride

A method is presented for the rapid extraction of proteins from Drosophila tissues. This method involves lysis of embryos in high concentrations of guanidine hydrochloride, followed by ultracentrifugation in a guanidine hydrochloride step gradient. Several membrane-associated antigens, including Notch and the β subunit of PS integrin are enriched in this preparation. The quantity of the proteoglycan, DROP-1, obtained from Drosophila eggs and testes was also greatly improved by the guanidine hydrochloride extraction method. This method should prove useful in the isolation and characterization of many Drosophila antigens, particularly those associated with cell membranes.

Comparison Between Catalytic Activity and Activity Changes During Denaturation by Guanidine Hydrochloride of Enzymes in Crystalline State and in Solution

The catalytic activity and activity changes during denaturation by guanidine hydrochloride of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and alpha-chymotrypsin in crystalline state and in solution have been compared. The catalytic activities are lower in crystalline state than in solution. Enzymes in crystalline state are more stable than in solution during denaturation by guanidine hydrochloride. Ammonium sulfate has different effects on catalytic activities of different enzymes and shows protection on all enzymes studied during denaturation by guanidine hydrochloride. The protection is more obvious at high concentrations of guanidine hydrochloride than at low concentrations. It is suggested that the flexibility or mobility of enzyme is required for the catalytic activity and related to the stability of enzymes. Enzymes with less flexibility or mobility are more stable.

Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and chemical characterization of the S layer of a Pseudomonas-like bacterium

Sections and freeze-fractured preparations showed an S layer on the surface of Pseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 +/- 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50 degrees C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50 degrees C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.

Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain.

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.

Refolding of ribonuclease in the presence and absence of ammonium sulfate pulses. Comparison between experiments and simulations.

Experiments have been carried out on ribonuclease A in which refolding in high concentrations of guanidine hydrochloride is either preceded or not preceded by a short ammonium sulfate pulse. Application of the pulse causes the rapid formation of the nativelike intermediate, and the effect of this pulse was determined by using three different methods for monitoring the subsequent refolding reaction: direct absorbance, direct fluorescence, and a double-jump fluorescence unfolding assay which is specific for the isomerization of proline-93. The effect of the pulse is quite different depending on the method of detection. With absorbance detection, the pulse causes a large reduction in the refolding amplitude with no change in the kinetics of the decay curve, while with the fluorescence unfolding assay, the pulse causes no change in the refolding amplitude but produces a large acceleration in the decay kinetics. The results with direct fluorescence are intermediate with some reduction seen in the refolding amplitude and some acceleration in the decay kinetics. The results of these experiments are simulated by using the simple model of Lin and Brandts (1984) [Lin, L.-N., & Brandts, J. F. (1984) Biochemistry 23, 5713] in which proline-93 must be in the correct cis configuration before folding to the native or nativelike state can occur. In all cases, the simulations accurately predict the experimental results for all three methods of detection, without any adjustment of parameter values from those published earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

The subunit structure and stability of conglutin δ, a sulphurrich protein from the seeds of Lupinus angustifolius L.

AbstractThe oligomeric structure and stability of conglutin δ, the 2S sulphur‐rich lupin seed protein, has been studied. Molecular weights were determined by sedimentation equilibrium methods in the analytical ultracentrifuge. Conglutin δ2 (Mr 14 000, 2S), the most abundant form, was composed of one large (˜9600) and one small polypeptide chain linked by disulphide bonds. The minor oligomeric form, conglutin δ1 (Mr 28 000, 2.8S), was a disulphide‐linked dimer of conglutin δ2. Each form was capable of reversible association and at low ionic strength (neutral pH), the conglutin δ1 momomer associated to a homogeneous dimer (Mr 56 000, 4.1S). Calculated frictional ratios (f/f0 ˜ 1.28–1.49) suggested that the three different forms were asymmetric. Spectral studies (ORD/CD) showed that conglutins δ1 and δ2 were rich in alpha‐helix (˜38%) unlike the major 7S and 11S legume seed storage proteins. The helical structure was unusually stable both to heat and chemical denaturants; below 60°C (at neutral pH) the helix remained unaffected and only partial denaturation occurred at higher temperatures. Nevertheless, complete denaturation was achieved (at 20°C) in high concentrations of guanidine hydrochloride (greater than 6.5 M). The stability was due to the presence of disulphide cross‐links; with the addition of reducing agent, as little as 1 M guanidine hydrochloride (GuHCl) was sufficient for denaturation of the helical structure. Titration experiments showed a single buried tyrosine (pK 11.4) which could be exposed (pK 10.2) in 6 M GuHCl.

Physical nature of the phase transition in globular proteins: Calorimetric study of human α-lactalbumin

The guanidine hydrochloride-induced unfolding of human α-lactalbumin has been studied by isothermal calorimetry. It has been shown that a cooperative transition takes place only in the concentration interval of the denaturant between 0.3 and 2 mol · 1 −1. The cooperative transition coincides with the transition detected by circular dichroism in the near-ultraviolet region which reflects the destruction of the specific environment of aromatic side groups. According to scanning calorimetric investigations, the transition disappears in the acid form of the protein where circular dichroism of aromatic side groups is practically absent. At higher concentrations of guanidine hydrochloride, where destruction of the secondary structure and un- folding of the chain are observed, there is no cooperative heat absorption.

Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains.

The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant. The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated. This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit. The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification. The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride.

LIGHT‐INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light‐induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR570) and M412‐intermediate, but decreased the decay rate of the M412‐intermediate. Light‐induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light‐induced proton transport. The role of amino group(s) in light‐induced proton release and transport through the purple membrane is discussed.

Solubilized Bone Morphogenetic Protein (BMP) from Mouse Osteosarcoma and Rat Demineralized Bone Matrix

A selection of proteins including bone morphogenetic protein (BMP) was extracted in a disaggregated form from Dunn osteosarcoma or rat demineralized bone matrix by 4M guanidine hydrochloride (GuHCl) solution without losing its biological activity. The GuHCl extracts of Dunn osteosarcoma were divied into 4 different fractions by cesium chloride (CsCl) density gradients. Under a dissociative condition, the highest new bone yield was obtained in the low dense top one-third fraction, and BMP acitivity declined with increase in the density of each fraction. No BMP potential was observed in the surface-gel fraction under dissociative conditions. Under an associative condition (low GuHCl concentrations), BMP activity appears in the surface-gel fraction, while under a dissociative condition (high concentrations of GuHCl) BMP appears in the fraction below the surface gel. These facts suggest that under associative conditions, BMP aggregates with other low dense proteins in the surface-gel fraction and that this may be the state of aggregation of BMP in cells and matrix in nature. Present observations support the assumption that BMP is a relatively low density protein and excludes the idea of BMP activity in the collagen molecule, per se. A specific protein, with an apparent molecular weight of 63,000 daltons, is present in all fractions that exhibit BMP activity, and absent in fractions that do not exhibit this activity. BMP is not species-specific; rat BMP induces bone formation in mice. CsCl density-gradient centrifugation is an efficient tool for further purification and isolation of BMP.

Altered activity of bacteriorhodopsin in high concentrations of guanidine hydrochloride.

Bacteriorhodopsin from Halobacterium halobium was stable and active in high concentrations of guanidine hydrochloride. However the decay of the intermediate with absorption maximum at 407 nm and the uptake of protons from the medium were remarkably delayed. At alkaline pH values, all the molecules of bacteriohodopsin existed as the 407 nm intermediate in the light, and one proton per bacteriorhodopsin molecule was released.

Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.

The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 25 degrees. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn.HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast- and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or urea-unfolded RNase A is the same as that in the heat-unfolded protein at pH 2. Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated Gdn.HCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn.HCl at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.

Characterization of a major allergen (cod): Observations on effect of denaturation on the allergenic activity

An allergenically active protein isolated from cod white muscle albumin and designated DS 22 was subjected to denaturation experiments by 8M urea/β-mercaptoethanol and high concentrations of guanidine hydrochloride. The allergenic activity and antigenicity of DS 22 were maintained after reduction and alkylation by iodoacetamide. The randomly folded material apparently recovered most of its native conformation by spontaneous reoxidation through dialysis against water. This was indicated by the recovery of the molecular immunopotency, ultraviolet spectral profiles, and electrophoretic characteristics. The findings suggest that the allergenic activity of DS 22 is not directly determined by the stereochemical configuration of the protein molecules but by the sequence of certain amino acids.

A structural subunit molecular weight of urease

The feasibility of determining protein molecular weights in high concentrations of guanidine hydrochloride by ultracentrifugation has been investigated. Ribonuclease was used as a nondissociating protein, and several modifications of the sedimentation equilibrium method were employed. An investigation of urease by these methods produced evidence for a structural subunit of 83300 molecular weight.

Combinations of modified serum γ-globulin with other proteins

Addition of serum γ-globulin exposed to high concentrations of guanidine hydrochloride for several hours to certain proteins results in formation of insoluble and soluble combinations of the modified serum γ-globulin with the other proteins. These combinations are formed under conditions where only the γ-globulin has been denatured. Effects of various experimental conditions on the formation of combinations have been evaluated. Egg albumin, serum albumin, and casein show a much smaller tendency to form combinations under comparable conditions than does γ-globulin. Combinations are readily obtained between modified γ-globulin and casein or native γ-globulin, but not between modified γ-globulin and serum albumin, egg albumin, or urease. Coupling of serum albumin or casein with diazotized sulfanilic acid or aniline markedly increases the extent of combination with the modified γ-globulin. At least two types of combinations are formed between the modified serum γ-globulin and serum albumin coupled with p-diazobenzenesulfonic acid. The insoluble combinations consist essentially of a matrix of γ-globulin from which the bulk of the combined azoalbumin can be extracted with alkyl or aryl sulfonates. The soluble combinations formed between modified γ-globulin and azoalbumin as well as some of the azoalbumin in the insoluble combinations cannot be dissociated under much more drastic conditions as evaluated by electrophoretic analyses. In electrophoresis the soluble combinations migrate as a single peak with mobility intermediate between the azoprotein and native γ-globulin.