Studies by UV spectroscopy of thermal denaturation of β-lactoglobulin in urea and alkylurea solutions

Poklar, Nataša; Vesnaver, Gorazd; Lapanje, Savo

doi:10.1016/0301-4622(93)85032-d

Cited by 33 publications

(35 citation statements)

References 18 publications

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“…Although the higher alkyl substitution of urea significantly lowers the concentration of urea needed to denature tr-ctg A, this does not mean that alkylureas are stronger denaturants than urea, because, as our results show, the protein final state in alkylurea solutions cannot be considered to be the same as in the urea solution (Warren and Gordon, 1976;Lapanje et al, 1981;Poklar et al, 1993).…”

Section: Discussionmentioning

confidence: 59%

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

1994

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The solvent denaturation of alpha-chymotrypsinogen (alpha-ctg A) in aqueous solution of urea, methyl-, N,N'-dimethyl-, ethyl-, propyl- and butylurea was studied by fluorescence measurements. Data were analyzed on the assumption of a two-state approximation to obtain the apparent equilibrium constant, Ku and the apparent Gibbs free energy of transition delta G0u. It has been observed that alkyl-substitution of urea significantly lowers the denaturant concentration needed to denature alpha-ctg A at 25 degrees C. Denaturation was accompanied by the red shift of emission maxima, the increase of the half-width of the fluorescence spectra, the increase of the fluorescence intensity, and the decrease of the fluorescence polarization. The differences of these fluorescence parameters observed for alpha-ctg A in alkylureas and urea can be ascribed to different unfolded states of the protein in different denaturant solutions. Minor differences in the extent of unfolding were confirmed by size-exclusion chromatography.

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Section: Discussionmentioning

confidence: 59%

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

1994

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“…An increased stability of proteins in the presence of sugar excipients has been shown to be a general phenomenon for many sugar/protein combinations (Timasheff, 1998). Although protein stability decreases in the presence of urea (Poklar et al, 1993) and guanidine hydrochloride (von Hippel and Wong, 1965), it increases in the presence of sugars, including sucrose (Lee and Timasheff, 1981) and trehalose (Xie and Timasheff, 1997b). The mechanism for stabilization of proteins in the presence of sugars is thought to be the result of the sugar being preferentially excluded from the protein surface (Timasheff, 1998).…”

Section: Discussionmentioning

confidence: 99%

Reduced protein adsorption at solid interfaces by sugar excipients

Wendorf

Radke

Blanch

2004

Biotech & Bioengineering

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Sugar excipients are shown to reduce the adsorption of ribonuclease A, bovine serum albumin, and hen egg white lysozyme at the liquid-solid interface. The amount of protein adsorbed decreased as the concentration of the sugar increased. At the same sugar concentration, the ability of sugars to reduce protein adsorption followed the trend: trisaccharides > disaccharides > 6-carbon polyols > monosaccharides. This trend in adsorbed protein amounts among sugars was explained by stabilization of the protein native state in solution by the sugar excipients. The heat of solution of the amorphous saccharide was found to correlate with the amount of protein adsorbed.

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“…The protein concentration at UV experiments was 0.25 mgm1-1. UV-melting curves were analyzed by assuming a two-state transition to obtain the apparent thermodynamic quantities of interest, the apparent equilibrium constants K2pp, the apparent standard Gibbs free energy Adaapp, the apparent standard enthalpy AdH~ the apparent standard entropy AdS~ 'app, and the apparent standard change in heat capacity AaC~ '~pp, following the procedure described in detail elsewhere (Poklar et al, 1993). …”

Section: Uv Spectroscopymentioning

confidence: 99%

“…The relative error in determination of all thermodynamic quantities at 25~ is estimated to be • It is well known that thermodynamic stability of small globular proteins decreases with increasing urea concentration (Makhatadze and Privalov, 1992;Gopal and Ahluwalia, 1991). Unfortunately, there are no literature data on the thermal stability of globular proteins in the presence of alkylureas except those of fl-lactoglobulin (Poklar et al, 1993).…”

Section: Effect Of Different Ureas On Thermal Stability Of O~-ctg a Omentioning

confidence: 99%

“…thermodynamic quantities of denaturation using the procedure described previously (Poklar et al, 1993). Table III presents the temperature of transition Ta uv and apparent standard enthalpy of denaturation AdH~ of O~-ctg A obtained at different pH, at different concentrations of added NaC1, and at different concentrations of various ureas.…”

Section: Effect Of Added Naci and Ph On Thermal Stability Of C~-ctg Amentioning

confidence: 99%

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Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

1995

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The effects of pH, urea, and alkylureas on the thermal stability of alpha-chymotrypsinogen A (alpha-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition of alpha-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25 degrees C were calculated. Comparison of these values shows that at 25 degrees C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein-molecules. At pH lower than 2.0, alpha-ctg A still exists in a very stable form, probably the acid-denatured from (A-form).

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Studies by UV spectroscopy of thermal denaturation of β-lactoglobulin in urea and alkylurea solutions

Cited by 33 publications

References 18 publications

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

Reduced protein adsorption at solid interfaces by sugar excipients

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

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