Course Of Denaturation Research Articles

Region-Specific Double Denaturation of Human Serum Albumin: Combined Effects of Temperature and GnHCl on Structural and Dynamical Responses.

In this work, we have investigated the effects of denaturing agents, guanidine hydrochloride (GnHCl) and temperature, on the overall structure, domain-I, and domain-III of human serum albumin (HSA) using circular dichroism (CD) spectroscopy and steady-state, time-resolved fluorescence spectroscopy. We have tagged Cys-34 of HSA, located at domain-I, using N-(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide and Tyr-411 of HSA, located at domain-III, using p-nitrophenyl coumarin ester, for this purpose. The CD spectroscopy studies reveal the overall denaturation of the protein. The denaturation follows the expected direction in which the protein is denatured with an increase in the concentration of GnHCl or temperature. The α-helicity of the native state of HSA was found to be 64.2%, and the minimum value of α-helicity was found to be 14.8% in the presence of 6 M GnHCl at room temperature. Steady-state emission studies were carried out on domain-I and domain-III of the protein using site-specific fluorescent tags. The degree of folding of the two domains at different combinations of temperature and GnHCl concentration was calculated and was found to follow a slightly different course of denaturation. Solvation dynamics was found to be quite different for these two domains. The domain-I of HSA has a maximum solvation time of 0.39 ns, and the solvation time tends to decrease with the action of either temperature or GnHCl. On the other hand, the domain-III of HSA showed a much higher solvation time (1.42 ns) and does not show any regular change at higher temperatures or in the presence of GnHCl. This difference could be attributed to the different microenvironment inside the protein cores of the two domains.

The fluorescence detected guanidine hydrochloride equilibrium denaturation of wild-type staphylococcal nuclease does not fit a three-state unfolding model

A three-state equilibrium unfolding of a protein can be difficult to detect if two of the states fail to differ in some easily measurable way. It has been unclear whether staphylococcal nuclease unfolds in a two-state fashion, with only the native and denatured states significantly populated at equilibrium, or in a three-state manner, with a well-populated intermediate. Since equilibrium unfolding experiments are commonly used to determine protein stability and the course of denaturation are followed by changes in the fluorescence which has difficulty in distinguishing various states, this is a potential problem for many proteins. Over the course of twenty years we have performed more than one hundred guanidine hydrochloride equilibrium denaturations of wild-type staphylococcal nuclease; to our knowledge, a number of denaturations unrivaled in any other protein system. A careful examination of the data from these experiments shows no sign of the behavior predicted by a three-state unfolding model. Specifically, a three-state unfolding should introduce a slight, but characteristic, non-linearity to the plot of stability versus denaturant concentration. The average residuals from this large number of repeated experiments do not show the predicted behavior, casting considerable doubt on the likelihood of a three-state unfolding for the wild-type protein. The methods used for analysis here could be applied to other protein systems to distinguish a two-state from a three-state denaturation.

Stability of hen egg white lysozyme during denaturation is enhanced by pretreatment with supercritical carbon dioxide

Supercritical carbon dioxide (SC-CO(2)) has been successfully employed in a variety of applications due to its numerous advantages. Despite extensive investigations on the relationship between the activity of enzymes treated with supercritical fluids and supercritical operating conditions, there are no experimental studies that have addressed the effects of supercritical pretreatment on enzyme denaturation. In this study, we have explored the impact of SC-CO(2) pretreatment on the activity and stability of hen egg-white lysozyme during its course of denaturation. Our data indicated no noticeable enhancement in enzyme activity and stability in the presence of SC-CO(2) pretreatment for lysozyme samples denatured in 8 M urea at 50 degrees C and pH 6.2. However, SC-CO(2) pretreated lysozyme samples in 0.067 M phosphate buffer containing dithiothreitol (DTT) (0.1 M DTT, pH 6.2, 25 degrees C or 0.01 M DTT, pH 6.2, 50 degrees C) at 2500 psi and 50 degrees C had better residual activity relative to samples that were not pretreated. In addition, when denaturing at 65 degrees C and pH 9.0, the pretreatment in SC-CO(2) at 2500 psi and 50 degrees C resulted in the best stability of lysozyme. The result of this study may provide supporting evidence that supercritical fluids serve as potential media for enhancing the activity of enzymes used in a variety of biochemical applications.

Pyrenemethyl ara-uridine-2'-carbamate: a strong interstrand excimer in the major groove of a DNA duplex.

The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA-DNA duplexes, the destabilization is considerably reduced. The pyrenemethyl derivative also shows remarkable spectral properties: a "reversed" absorbance change for pyrene at 350 nm in the course of denaturation of the DNA duplex, as compared to the change seen in the nucleotide absorbance at 260 nm. This derivatization also causes pronounced sequence-dependent excimer formation in the major groove.

Inactivation and conformational changes of lactate dehydrogenase from porcine heart in sodium dodecyl sulfate solutions

The inactivation and conformational changes of porcine heart lactate dehydrogenase (LDH) have been studied in sodium dodecyl sulfate (SDS) solutions. Increasing SDS concentration led to a quick and concentration-dependent inhibition of the enzyme, with complete inactivation within 5 min in the presence of 1.0 mM SDS. Meanwhile, fluorescence emission and circular dichroism spectra were used to follow the conformational changes of the enzyme during this process, concurrently showing that SDS less than 1.0 mM induced only limited conformational changes to LDH. The above results are in accordance with the suggestion by Tsou (Trends Biochem. Sci. 11 (1986) 427; Science 262 (1993) 380) that the active site usually be more flexible than the enzyme molecule as a whole. Furthermore, the results of polyacrylamide gel electrophoresis (PAGE) implied that unfolding intermediates were presented in the above process. When the SDS concentration used to treat LDH was increased, the bands of native enzyme on native PAGE faded and finally almost disappeared. Meanwhile, multiple bands with lower mobility but no activity emerged behind and enhanced correspondingly. Fast protein liquid chromatography indicated that dissociation occurred during the course of denaturation. The reasons for the above phenomena have been discussed. It was suggested that SDS, binding to LDH to form different LDH–SDS complexes, conferred an array of different unfolding states over the enzyme, and in turn resulted in the formation of the multiple bands on the native PAGE.

Estimation of Protein Secondary Structure from Circular Dichroism Spectra: Inclusion of Denatured Proteins with Native Proteins in the Analysis

We have expanded our reference set of proteins used in the estimation of protein secondary structure by CD spectroscopy from 29 to 37 proteins by including 3 additional globular proteins with known X-ray structure and 5 denatured proteins. We have also modified the self-consistent method for analyzing protein CD spectra, SELCON3, by including a new selection criterion developed by W. C. Johnson, Jr. (Proteins Struct. Funct. Genet. 35, 307–312, 1999). The secondary structure corresponding to the denatured proteins was approximated to be 90% unordered, owing to the spectral similarity of the denatured proteins and unordered structures. We examined the thermal denaturation of ribonuclease T1 by CD using both the original and expanded sets of reference proteins and obtained more consistent results with the expanded set. The expanded set of reference proteins will be helpful for the determination of protein secondary structure from protein CD spectra with higher reliability, especially of proteins with significant unordered structure content and/or in the course of denaturation.

Dynamic rheological behaviour and biochemical properties of rabbit skeletal actomyosin during storage at 0° C

AbstractThe relationship between the dynamic rheological properties of heat induced gels of actomyosin (natural actomyosin) and the denaturation of actin in actomyosin during storage without ATP at pH 6.0 and 0°C was investigated using biochemical and dynamic rheological measurements. The complex modulus of gels after heating actomyosin containing 0.5 or 1.5 M KCl (pH 6.0) at 80°C increased with increasing storage time. The dynamic rheological behaviour during heat gelation of actomyosin in 1.5 M KCl indicated that the first rheological transition peak in the 50–53°C range induced by the presence of F‐actin gradually disappeared with increasing storage time. However, in 0.5 M KCl, this transition peak clearly remained even after 15 days. The time course of denaturation of actin in actomyosin treated with 1.5 M KCl at pH 6.0 showed an increase in the percent denaturation after the storage was started, and about 100% of the actin became denatured after 21 days. In the case of 0.5 M KCl, unlike 1.5 M, the denaturation of actin occurred quickly within the first 5 days and then did not proceed. A sigmoidal relationship was found between the percent denaturation of actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.5 and 1.0 M. The data indicated that the change in the property of actin in actomyosin during storage at low temperature exerts a great influence on the viscoelasticity of heat‐induced gels of actomyosin.

Intrinsic stability and extrinsic stabilization of creatinase from Pseudomonas putida.

Creatinase (creatine amidinohydrolase, EC 3.5.3.3), a homodimer of 45 kDa subunit molecular mass, shows only limited functional stability, and is inaccessible to reconstitution after preceding deactivation, denaturation and dissociation. The enzyme has been characterized regarding its native and denatured states. Studying its unfolding characteristics in the presence of "extrinsic factors", such as DTE, BSA and glycerol, it was possible to define solvent conditions where the stability of the enzyme is significantly improved. Apart from protecting essential thiol groups and charge screening effects, the stabilization is caused mainly by preferential solvation. In the presence of 20% (w/v) glycerol, the kinetic analysis of the time course of denaturation indicates that a partially active folding intermediate, rather than the whole molecule, is involved in the stabilization. The mixed solvent improves the thermal stability, as well as the stability toward GdmCl and urea.

The effect of some medical treatments of thalassemia on the red blood cells

The Mossbauer spectroscopy (MS) and circular dichroism (CD) measurements have been used to investigate the effect of some medical treatments on the red blood cells (RBCs) of the patients with HbH discase and β-thalassemia (Thal.) major, respectively. The results indicate that both splenectomy and treatment with myleran are effective to alleviate the symptoms of anemia for some patients, but both of them are different in the effect on the RBCs of the patients. On the basis of the results, a hypothesis on the course of denaturation in hemoglobin (Hb) of the patients is proposed.

Denaturant-gradient chromatography for the study of protein denaturation: Principle and procedure

The particle volume of a protein which represents the expansion of the molecular chain is a useful probe to measure its conformation change in solution. To study the details of the conformation transition, we will report a novel device to trace the particle-volume change against increasing denaturant concentration. According to the denaturant gradient in the eluant of high-performance gel-permeation chromatography, information concerning the elution volume of proteins in the course of denaturation can be successively accessed. When a protein is injected into a denaturant solution, its effective molecular volume increases by the unfolding of a native compact structure, and the elution peak goes forward to separate from that of the native protein. Typical elution patterns were computer-simulated in an extended algorithm of the plate theory. The kinetic feature of denaturation and the effect of the denaturant gradient are discussed. The denaturation schemes of lysozyme, cytochrome c, and phosphoglycerate kinase were studied.

Ageing of α-chymotrypsin: Cannibalistic and hydroxide ion reactions

The loss of catalytic activity (irreversible denaturation) of alpha-chymotrypsin (EC 3.4.21.1) as a function of pH between 6 and 13 is described. Around neutrality the irreversible denaturation is second order with respect to enzyme. At high pH, the irreversible denaturation is first order with respect to enzyme and also with respect to hydroxide ion. The first- or second-order dependency on enzyme concentration has been shown both by the time course of denaturation and by the effect of enzyme concentration on the rate of denaturation. The first-order dependency on hydroxide ion was shown by a plot of the logarithm of the rate constant vs. pH. These enzyme inactivations are peptide bond cleavages at all pH values. From the kinetics, the irreversible denaturation can be described as a cannibalistic reaction of two enzyme molecules around neutrality or as a reaction of one enzyme molecule plus one hydroxide ion at high pH.

Effect of guanidine hydrochloride on the hydrodynamic and thermodynamic properties of human apolipoprotein A-I in solution.

To further define its properties in solution, the course of denaturation of apolipoprotein A-I (apo-A-I) was studied as a function of various concentrations of guanidine hydrochloride (GdmCl) using the techniques of circular dichroism, ultraviolet difference spectroscopy, analytical ultracentrifugation, viscometry, and densitometry. At low molarities of GdmCl (0 to 0.4 M), apoA-I exhibited anomalous ellipticity changes which were dependent upon apo-A-I concentration; from ultracentrifugal studies, these changes were interpreted as related to the dissociation of protein oligomers into a new equilibrium attended by a preferential interaction with GdmCl (0.07 g/g of apo-A-I and an increase in molar volume, 197 ml). On the other hand, the frictional (1.68) and axial (5.7) ratios were similar to those in the native state. At molarities of GdmCl between 0.4 M and 6 M, apo-A-I dissociated into monomers by a cooperative denaturation process which was independent of protein concentration: both dissociation and denaturation were thermodynamically reversible with an apparent free energy of stabilization, = 3.70 kcal/mol. Between 2 and 6 M GdmC1, changes in the conformation, intrinsic viscosity, and volume of apo-A-I were noted as assessed by spectroscopic, viscometric, and partial specific volume measurements. These changes appeared related to the number of moles of GdmCl bound to apo-A-I (at 2 M GdmC1, 63 mol; at 6 M, 181 mol).

Further studies of the membrane-bound iron-sulfur proteins and P700 in a photosystem I subchloroplast particle

The denaturation by urea-ferricyanide of the membrane-bound iron-sulfur proteins in a spinach photosystem I particle was investigated further. We observed the following results: (i) During the course of the denaturation, the conversion of labile sulfide to zerovalence sulfur proceeds with an equivalent loss of nonheme iron. (ii) Particles depleted of nonheme iron and labile sulfide lack electron spin resonance Centers A and B after chemical or photochemical reduction. (iii) The inorganic sulfide, previously shown to be regenerated in the inactivated particle by dithiothreitol (J. H. Golbeck, S. Lien, and A. San Pietro, 1977, Arch. Biochem. Biophys. 178 , 140–150), becomes bound to the particle only if inorganic iron is present. (iv) This binding to the particle is probably in the form of an artificial iron-sulfur cluster, inasmuch as electron spin resonance and photochemical assays failed to reveal recovery of activity. (v) During the course of urea-ferricyanide denaturation, the relationship between the remaining bound iron-sulfur protein and the flash-induced P700 oxidation proved to be strictly linear. This implies that all 10–12 Fe-S pairs which are present in each photosystem I unit show equal sensitivity to oxidation by urea-ferricyanide. Consequently, we suggest the possibility that the bound iron-sulfur centers are located on a single protein.

Spin-label studies of tropomyosin

Studies are reported on nitroxide spin-labeled tropomyosin. The labels attach to sulfhydryl groups and to amino groups. The amino spins are highly mobile, the sulfhydryl much less so. Spin count studies show an average of approximately 0.5 labeled sulfhydryl/tropomyosin molecule and only approximately 0.15 labeled amino group/molecule. The spectra are used tostudy the denaturation of tropomyosin by guanidine hydrochloride. The information obtained reveals the course of denaturation at sites near the sulfhydryl group. It is found that these sites are more susceptible to guanidine than the bulk of the molecule; denaturation at the sulfhydryl sites is complete by 1.5 M guanidine, whereas optical studies indicate the molecule as a whole is not completely denatured until the concentration reaches 3.5 M. Spectra are also shown of tropomyosin fibers oriented variously with respect to the applied magnetic field. Strong orientation effects are seen and these indicate that the sulfhydryl-attached spins (but not the amino-attached spins) have a definite orientation in the fiber. Interpretation of the spectra reveals that the normal to the nitroxide plane is inclined to the fiber axis at an angle of 50 degrees. Circular dichroism studies in the tyrosine region also reveal drastic changes with guanidine denaturation, confirming the idea that denaturation produces pronounced increase in mobility at the beta carbon (as in the sulfhydryl casey). A strong negative band existing only in helical tropomyosin at pH's where the tyrosines are uncharged appears to be due to interaction of tyrosines with the helical backbone, whereas the appearance of a strong positive CD band at 250 nm at high pH (approximately11) seems to be ascribable to interaction between the charged phenolic groups and the dissymmetric backbone alpha-carbon atom.

Renaturation of soluble collagen: III. Reorganization of native collagen molecules from completely separated units

Acid-soluble collagen in citrate buffer (pH 3.7) was completely denatured at 38 °C. and renatured by temperature fluctuation between 4 ° and 22 °C. The renaturation process was observed by measurements of light scattering, viscosity, and optical rotation. In this case a higher degree of renaturation was obtained than by isothermal renaturation at 4 °C. In order to isolate the regenerated native molecules, the residual denatured material was removed by treatment with pepsin or trypsin. The enzyme-resistant fractions show the physical properties of native tropocollagen. They form well-structured long-spacing segments and native-type fibrils, and show the same or a similar course of denaturation, viscosity, and sedimentation constant. The yield of completely renatured material is about 50%.

Physicochemical Studies on the Reaction between Formaldehyde and DNA

The reaction between formaldehyde and phage T7 DNA has been studied by optical absorbance and sedimentation measurements. Through the course of denaturation, OD(200) and s(20, w) rise; after the attainment of full hyperchromicity the s(20, w) falls sharply, suggesting a decrease in molecular weight. Conditions in which formaldehyde causes cross-linking are defined. Some experimental applications of the denaturation technique are given. Evidence which suggests that preformed single-strand interruptions may exist in phage DNA is briefly discussed.

Denaturation of Hemoglobin.

In a recent series of papers by Anson and Mirsky, the claim is made that hemoglobin, in N/20 HCl, is completely denatured in 3 minutes at 0°C. These authors claim, further, that this denaturation may be reversed to the extent of 75% by simply neutralizing the acid added with an equivalent amount of NaOH containing a small amount of NaCN. These experiments on the reversal of hemoglobin denaturation are, in essential details, substantially the same as those published almost 3 years earlier by Wu and Lin. Wu and Lin, however, considered that they were dealing not with true denaturation but with a pseudo-denaturation which was peculiar to hemoglobin and dependent upon its prosthetic group. In order to test these divergent views, we repeated the experiments of Anson and Mirsky at temperatures which would really denature the protein. Instead of insolubility at the iso-electric point, we made use of precipitin reactions against a specific antiserum to follow the course of denaturation. Since true denaturation of a protein results in a gradual diminution of its ability to react with its antiserum, we may expect that any reversal of this denaturation would manifest itself by a return of its precipitinogenic properties. A 3% solution of ox carbon monoxide hemogloibn, prepared by the method previously described by Boor and Hektoen was used in these experiments. One volume of this solution was treated with 3 volumes of N/15 HCl and, after standing at room temperature for 10 minutes, the brown mixture was placed in a boiling water bath. At stated intervals, 4 cc. of this mixture was removed and neutralized by the addition of 1 cc. N/5 NaOH and 1/20 cc. N. NaCN.