Why Is Trehalose an Exceptional Protein Stabilizer?

Abstract

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...

Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

“…In this study, DSC and QELS have been used to assess both types of stability in solutions prepared under exactly the same conditions. While several researchers have investigated the effects of sugars on both structure 2,13 and solution stability, 6 this has not been done at the same time and under the same conditions before now. Therefore, this study allows us to directly compare the stabilities, at the same ionic strength and pH and establish if there is a correlation between the two.…”

“…1−7 Therapeutic proteins are produced and used for the treatment of various human diseases 8 such as insulin for the treatment of diabetes, 9 monoclonal antibodies for rheumatoid arthritis, 10 interferon-α for leukemia, 11 and interferon-β for multiple sclerosis. 12 Sugars are generally used as protein structure stabilizers, 2,13 since additional stability is conferred to a protein during lyophilization by the addition of the sugar. 14 Two hypotheses have been proposed to describe the stabilizing effect of sugars on proteins during lyophilization; one such hypothesis states that sugar acts as a water substituent and stabilizes proteins by forming hydrogen bonds at specific sites on the surface of the protein.…”

“…The stabilization of protein structure against thermal denaturation is referred to as its thermal or structural stability. Spectrophotometric techniques 13,29−31 and differential scanning calorimetry (DSC) 32,33 have been used to measure the effect of several additives on the thermal stability of proteins including sugars, 13,29,30,32 amino acids, 33 and salts. 31,32 These techniques have been used to measure the thermal transition temperature (T m ) and calorimetric enthalpy (ΔH) of protein solutions in the absence and presence of additives.…”

Analytical Methods for the Detection and Purification of ε-Poly-L-lysine for Studying Biopolymer Synthetases, and Bioelectroanalysis Methods for Its Functional Evaluation

“…It is noted that 1 g/L monomeric L-lysine did not decrease the k-value significantly. Also, no significant decrease in k was observed with 1 g/L of well-known stabilizers, glycerol, 39 trehalose, 40 and ammonium sulfate, which are usually added at much higher concentrations.…”

Preventing Aggregation of Recombinant Interferon beta-1b in Solution by Additives: Approach to an Albumin-Free Formulation

“…37 Trehalose has been reported to destabilize some proteins at high pH and to have pHdependent stabilizing effects. 22,38 This could help minimize aggregation using trehalose additive in optimized concentration at low pH in formulations by experimental design. In fact, protein hydrophobicity increases with decreasing pH due to the protonation of COO− groups.…”