Denaturation studies on bovine serum albumin–bile salt system:Bile salt stabilizes bovine serum albumin through hydrophobicity

Protein denaturation is under intensive research, since it leads to neurological disorders of severe consequences. Avoiding denaturation and stabilizing the proteins in their native state is of great importance,especially when proteins are used as drug molecules or vaccines. It is preferred to add pharmaceutical excipients in protein formulations to avoid denaturation and thereby stabilize them. The present study aimed at using bile salts(BSs), a group of well-known drug delivery systems, for stabilization of proteins.Bovine serum albumin(BSA) was taken as the model protein, whose association with two BSs, namely sodium cholate(Na C) and sodium deoxycholate(Na DC), was studied. Denaturation studies on the preformed BSA-BS systems were carried out under chemical and physical denaturation conditions. Urea was used as the chemical denaturant and BSA-BS systems were subjected to various temperature conditions to understand the thermal(physical) denaturation. With the denaturation conditions prescribed here,the data obtained is informative on the association of BSA-BS systems to be hydrophobic and this effect of hydrophobicity plays an important role in stabilizing the serum albumin in its native state under both chemical and thermal denaturation.

CALORIMETRIC STUDY OF COLD AND THERMAL DENATURATION OF BOVINE β-LACTOGLOBULIN B

The denaturational behaviour of bovine β-lactoglobulin B has been studied in solutions containing guanidine hydrochloride by differential scanning calorimetry. The experiments have shown that complete peaks of cold denaturation can be recorded also in high concentration of protein solutions. The cold denaturation and the renaturation of the protein are reproducible, but the thermal denaturation is irreversible. The activation energy of thermal denaturation calculated is about 285 kJ/mol.

OBSERVATION OF DNA PARTIAL DENATURATION BY ATOMIC FORCE MICROSCOPY

Atomic force microscopy was used to investigate the DNA-cetyltrimethylammonium bromide (CTAB) complexes adsorbed on highly ordered pyrolytic graphite (HOPG). These complexes, at low concentrations, can automatically spread out on the surface of HOPG. The DNA-CTAB complexes display a typically extended structure rather than a globular structure. Partially denaturated DNA produced by binding CTAB to DNA is directly observed by AFM with high resolution. The three-dimensional resolution of partially denaturated DNA obtained by AFM is not available by any other technique at present.

Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein Heterogeneity

Urea denatures proteins at different concentrations, depending on the experimental conditions and the protein. We in-vestigated the pressure-induced denaturation of bovine serum albumin (BSA) in the presence of subdenaturing concen-trations of urea based on a two-state equilibrium. Pressure-induced denaturation was enhanced at urea concentrations ([U]) of 3.5 M to 8.0 M, with the free energy of denaturation at atmospheric pressure ranging from +5.0 to –2.5 kJ/mol of BSA. The m values appeared to be biphasic, with m1 and m2 of 0.92 and 2.35 kJ mol–1?M–1, respectively. Plots of versus ln[U] yielded values of u, the apparent stoichiometric coefficient, of 1.68 and 6.67 mol of urea/mol of BSA for m1 and m2, respectively. These values were compared with the m and u values of other monomeric proteins reported in or calculated from the literature. The very low values of u systematically observed for proteins were suggestive of heterogeneity in the free energy of denaturation. Thus, a u value of 140 mol of urea/mol of BSA may indicate the existence of a heterogeneous molecular population with respect to the free energy of dena-turation.

Theory of cold denaturation of proteins

A new approach to the problem of cold denaturation is presented. It is based on solvent-induced effects operating on hydrophilic groups along the protein. These effects are stronger than the corresponding hydrophobic effects, and they operate on the hydrophilic groups which are plentiful than hydrophobic groups. It is shown that both heat and cold denaturation can be explained by these hydrophilic effects.

Denaturation of DNA in Ternary Mixed Solution of Water/Hydrophilic/Hydrophobic Organic Solvent

Denaturation was examined for the first time in a ternary mixed solution of water/hydrophilic/ hydrophobic organic solvent using λ-DNA and a plasmid as models. The absorbance of λ-DNA and the plasmid at 260 nm gradually increased for several days up to 1.68 and 1.38 times the initial values, respectively, in a water/acetonitrile/ethyl acetate (15:3:2, volume ratio) mixed solution, whereas there was little change in a water/acetonitrile (15:3, volume ratio) mixed solution. The plasmid treated with the ternary mixed solution was also examined with agarose gel electrophoresis. These experimental data indicated that λ-DNA changed from a double helix structure to a single helix structure and that the plasmid partially transformed to generate a denaturation bubble in the structure. The new idea of using the ternary mixed solution first enabled the interaction of the hydrophobic organic solvent (e.g., ethyl acetate) molecule with the double helical structure of DNA, leading to specific slow-proceeding denaturation.

Analysis of Lattice Size, Energy Density and Denaturation for a One-Dimensional DNA Model

There are several mechanical models to describe the DNA phenomenology. In this work the DNA denaturation is studied under thermodynamical and dynamical point of view using the well known Peyrard-Bishop model. The thermodynamics analysis using the transfer integral operator method is briefly reviewed. In particular, the lattice size is discussed and a conjecture about the minimum energy to denaturation is proposed. In terms of the dynamical aspects of the model, the equations of motion for the system are integrated and the results determine the energy density where the denatura- tion occurs. The behavior of the lattice near the phase transition is analyzed. The relation between the thermodynamical and dynamical results is discussed.

Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR

Conventional prenatal diagnosis relies on invasive chorionic biopsy or amniocentesis, which increases the risk of miscarriage, and is undertaken at 11-20 weeks gestation.1 The discovery of cell-free fetal DNA in maternal plasma has, however, offered a new strategy for non-invasive prenatal diagnosis.2 Cell-free fetal DNA in maternal plasma has been used for the determination of fetal gender3 and RHD status4 as well as testing certain monogenic diseases such as 13-thalassemia5 and cystic fibrosis.6 However,

DSC study of cold and heat denaturation processes of β-lactoglobulin A with guanidine hydrochloride

The cold and heat denaturations of bovine β-lactoglobuhn A (β-lg A) has been studied in solutions of guanidine hydrochloride (GuHCl) by differential scanning calorimelry (DSC) The experimental results are presented and discussed.It is shown that the number of protons bound by the monomeric molecules of β-lg A was unchanged before and after its heat denaturation below pH 3,and that the activation energy of the heat denaturation was depressed owing to the presence of GuHCl.In the solutions with 2.50 and 3.06 mol/L of GuHCl,both the cold and heat denat-urations of β-lg A were observed.In comparison with the heat denaturation,the activation energy of cold denaturation was far lower and the number of GuHCl molecules bound by the unfolded polypeptide chains after cold denaturation increased a lot.The absolute value of the enthalpy of cold denaturation was larger than that of heat denaturation It was found by the analysis that the contribution to the total denaturational enthalpy of conformational change

Effect of Native Disulfide Bonds on the Denaturation of Proteins in 6 mol/L Guanidinium Chloride——A Fourier Transformed Infrared Study

The unfolding of bovine serum albumin, lysozyme and ribonuclease A denatured in 6 mol/L GuHCl with their disulfide bridges intact and reduced have been compared by FTIR studies. The peak positions and heights in the deconvolved spectra of amide I bands of the above denatured proteins with native disulfide bonds show marked differences whereas those for the denatured proteins without disulfide linkages are closely similar. The above and other evidence suggest that denatured proteins with intact disulfides still have considerable ordered conformation even in 6 mol/L GuHCl.

DSC study of denaturation of β-lactoglobulin B

The denaturation of bovine β-lactoglobulin B (β-Lg B) has been studied in phosphate solutions with various concentrations of GuHCl with differential scanning calorimetry The experiments demonstrated that the presence of GuHCl made the β-Lg B undergo both cold denaturation and heat denaturation under the condition of a high concentration of the protein. The enthalpy changes of both kinds of denaturation exhibit opposite signs. Both the cold denaturation and the renaturation of the protein are reproducible, but its heat denaturation is irreversible. The cooperation among monomer molecules of the protein is involved in its heat denaturation The heat denaturation of the protein can be represented by the thermodynamic model Nc D→F. The activation energy of heat denaturation is 285 kJ/mol, which imples that the depression of temperature and enthalpy of heat denaturation of the P-Lg B does not result from decreasing considerably the activation energy by GuHCl As for the cold denaturation of the protein,

Calorimetric Study of Thermal Denaturation of Superoxide Dismutase

The thermal denaturation of superoxide dismutase (SOD) from bovine erythrocytes was studied at various pH values of different buffers and at various concentrations of solutions of two neutral salts by differential scanning calorimetry. The experiments performed indicate that the PIPES is a buffer non-coordinating with the SOD, and that the binding of the anions studied influences more or less the thermal denaturation of SOD, but the effect on the oxidation form of SOD is more apparent. A new conformer of SOD with lower thermostability was discovered by the experiments performed in different buffers at certain pH values higher than the isoelectric point of SOD, or at higher concentrations of neutral salt solutions. The new conformer may be converted irreversibly into the usual conformer with high thermostability during heating. Based on the thermodynamic parameters obtained in distilled water and by thermodynamic analysis using the Ooi’s model, it is revealed that the large enthalpy △Hdc contributed

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the dena- turation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.

DSC study of cold and heat denaturation of β-lactoglobulin A with urea

IN the field of thermodynamics of protein solutions, there are two kinds of denaturation induced by temperature changes. One of them is the commonest heat denaturation induced by temperature increasing and accompanied by a heat absorption and an increase of enthalpy and entropy. The other is cold denaturation, predicted by Brandts over thirty years ago: the compact ordered structure of protein molecules in the native state is induced into a

Conformational Changes at the Active Site of Creatine Kinase During Denaturation of Urea

It has been previously reported that during the denaturation of creatine kinase (ATP: creatine phosphotransferase EC 2.7.3.2) by guanidine hydrochloride, urea or SDS, inactivation occurs before noticeable conformational change of the enzyme molecule as a whole can be detected, and the inactivation rate constants of the enzyme in the above denaturant solutions are several orders of magnitude faster than those of

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 α-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.

Unfolding and Inactivation During Thermal Denaturation of Aminoacylase

Many researchers have compared inactivation with conformational changes of a number of enzymes during denaturation by guanidine hydrochloride and urea. The results obtained show that inactivation occurs before noticeable conformational change of the enzyme molecule as a whole can be detected. The inactivation rate constants

Unfolding and Inactivation of Green Crab (Scylla Serrata) Alkaline Phosphatase during Thermal Denaturation

Enzyme activity is strongly dependent on its conformational integrity. The present paper compares the inactivation and unfolding of green crab (Scylla serrata) alkaline phosphate during thermal denaturation. The results show that inactivation takes place before noticeable conformational changes. This is in general accord with the suggestion previously made by Tsou, indicating that the active site of multi metal enzymes is situated in a region more flexible than the molecules as a whole.

Comparison between conformational change and inactivation rates of aminoacylase during denaturation in urea solutions

The kinetic method of the substrate reaction in the presence of mactivator previously described by Tsou has been applied to the determination of inactivation rates of aminoacylase during denaturation in urea solutions. The protective effect of substrate on the inactivation of aminoacylase by urea has been investigated. Simultaneously, the comparison between conformational change and inactivation rates of enzyme in the urea solutions of different concentrations has been studied. Results obtained show that the inactivation rate constants of the enzyme are larger than the rate constants of conformational changes. The present results show that the active site of metal enzyme-aminoacylase is also located in a limited and flexible region of the molecule that is more sensitive to denaturants than the enzyme as a whole.

Heat Denaturation of Protein Structures and Chlorophyll States in PSII Membranes

Heat denaturation is an important technique in the study of the structure and function of photosynthetic proteins. Heat denaturation of photosystem II (PSII) membrane was studied using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and oxygen electrode. Complete loss of oxygen evolving activity of the PSII membrane was observed at temperatures below 45℃ . The decrease of excitonic interaction between chlorophyll molecules occurred more rapidly than the change of the protein secondary structure of the PSII membrane at temperatures above 45℃ . The results indicate that the protein secondary structure of the membrane proteins in PSII membranes is more stable than the excitonic interaction between chlorophyll molecules during heat denaturation.