Thermal Stability of Proteins in Aqueous Polyol Solutions:  Role of the Surface Tension of Water in the Stabilizing Effect of Polyols

Kaushik, Jai K.; Bhat, Rajiv

doi:10.1021/jp981119l

Cited by 207 publications

(115 citation statements)

References 50 publications

(99 reference statements)

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“…3 suggest a good correlation of the effect of the increased surface tension of trehalose solutions with the increase in the T m for all the proteins studied. Studies done by us earlier using a series of polyols (25) and carboxylic salts (26) also indicate a strong correlation of the surface tension effect with the thermal stability of proteins, suggesting an important role of water and the solvent environment in the stability of proteins.…”

Section: Resultsmentioning

confidence: 85%

“…Denaturation at pH 7 for the studied proteins was carried out in the presence of 1.5 M GdmCl. several other osmolytes like sarcosine (29) and polyols (25) have also been observed to decrease the apparent heat capacity of denaturation of globular proteins. Neutral salts, including carboxylic acid salts (26), which increase the thermal stability of proteins, also lead to a decrease in the denaturation heat capacity of several proteins just like osmolytes.…”

Section: Resultsmentioning

confidence: 99%

“…We used all the experimental data points obtained in a transition reaction to fit Equation 2, as follows, and described in detail elsewhere (25,26),…”

Section: Methodsmentioning

confidence: 99%

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Why Is Trehalose an Exceptional Protein Stabilizer?

Kaushik¹,

Bhat

2003

Journal of Biological Chemistry

Self Cite

545

246

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Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 M trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18°C and Gibbs free energy by 4.8 kcal mol ؊1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (⌬C p ) in trehalose solutions for all the studied proteins. An increase in the ⌬G and a decrease in the ⌬C p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (⌬G versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 M concentration 4 -7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.Sugars have been known to protect proteins against loss of activity (1, 2), chemical (3, 4), and thermal denaturation (5-9). Among several sugars, ␣,␣-trehalose (␣-D-glucopyranosyl(131)-␣-D-glucopyranoside) has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation (10, 11). It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12, 13). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13, 14).Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17). Several studies carried out by Timasheff and coworkers (9,18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabil...

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Why Is Trehalose an Exceptional Protein Stabilizer?

Kaushik¹,

Bhat

2003

Journal of Biological Chemistry

Self Cite

545

246

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show abstract

“…This reduction of protein volume is accompanied by a concomitant reduction in the hydrophobicity of the protein surface, thus reducing the tendency for protein-protein self association [13]. Kaushik and Bhat [98] have pointed out to another possible stabilization mechanism that has to be considered. They explained the stabilization effect of certain polyols and sugars being related to the surface tension increase (for more details see [98]).…”

Section: Effect Of Various Pharmaceutical Excipients (Sucrose and Glymentioning

confidence: 99%

“…Kaushik and Bhat [98] have pointed out to another possible stabilization mechanism that has to be considered. They explained the stabilization effect of certain polyols and sugars being related to the surface tension increase (for more details see [98]). …”

Section: Effect Of Various Pharmaceutical Excipients (Sucrose and Glymentioning

confidence: 99%

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

Brun¹,

Frieß²,

Schultz-Fademrecht

et al. 2009

Biotechnology Journal

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The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein‐protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B22), a parameter of solution non‐ideality, which is obtained using self‐interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B22 was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B22 is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B22 decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein‐protein interactions. The B22 parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting‐in/salting‐out effect. It is also shown that B22 correlates with protein solubility as well as physical protein stability.

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Osmolyte-induced changes in protein conformational equilibria

et al. 2000

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Examining solute‐induced changes in protein conformational equilibria is a long‐standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute‐induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte‐induced changes in the stabilities of the A and N states of yeast iso‐1‐ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A‐state formation from acid‐denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water–protein and osmolyte–protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water–protein and solute–protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte‐induced protein stabilization from the literature, and by comparison with other analyses of solute‐induced changes in conformational equilibria. © 2000 John Wiley & Sons, Inc. Biopoly 53: 293–307, 2000

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Thermal Stability of Proteins in Aqueous Polyol Solutions: Role of the Surface Tension of Water in the Stabilizing Effect of Polyols

Cited by 207 publications

References 50 publications

Why Is Trehalose an Exceptional Protein Stabilizer?

Why Is Trehalose an Exceptional Protein Stabilizer?

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

Osmolyte-induced changes in protein conformational equilibria

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