Additive Effects of Alcohols and Polyols on Thermostability of Pepper Leaf Extracts

Chemical chaperones (CC) are plant stress-related compounds that can stabilize protein structure in adverse environments. Modes of action are thought to involve hydrogen bonding, primarily with the solvent, and hydrophobic stabilization of the protein core. The objective of this study was to determine structure–function relationships between CC (and structurally related compounds) and thermal stability of pepper (Capsicum annuum L.) leaf proteins. Both polarity [based on log Kow (the oil–water partition coefficient)] and capacity for hydrogen bonding (based on the number of OH groups) contributed to whether low-molecular-weight alcohols and polyols stabilized or destabilized proteins at elevated temperatures. Thermal stability increased with increasing number of OH groups at a fixed number of carbon atoms per molecule. Conversely, thermal stability decreased with increasing number of carbon atoms with a fixed number of OH groups. When CC solution concentrations were adjusted to the same concentration of OH groups (1.51 · 10 OH groups per milliliter), protein thermal stability increased with increasing CC polarity. Mixtures of different CC had additive effects on increasing protein thermostability, but mixtures of stabilizing (mannitol) and destabilizing (methanol) compounds negated each other. As a strategy for increasing plant thermotolerance, identification and removal of destabilizing compounds should be equally effective as increasing levels of stabilizers in protecting protein conformation at elevated temperatures. Two primary focal points in abiotic stress tolerance are heat shock proteins and compatible solutes. Compatible solutes have been implicated in resistance to water deficit, salt, and temperature stresses and can accumulate to high concentrations without disrupting protein structure. Chemical chaperones (CC), including sugars, polyols, amino acids, and methylamines, are a subset of compatible solutes capable of stabilizing protein structure. Some of the more extensively studied compounds with CC activity include mannitol (Ruijter et al., 2003), glycerol (Kim and Lee, 1993), trehalose (Kaushik and Bhat, 2003), maltose (Kaplan and Guy, 2004), sucrose (Millard et al., 2003), glycine betaine (Chow et al., 2001), and trimethylamine oxide (Bennion and Daggett, 2004). A number of organisms accumulate CC to stabilize protein structure during stresses that promote denaturation (Managbanag and Torzilli, 2002). Trehalose plays a significant role in acquisition of heat tolerance in Saccharomyces cerevisiaeMeyen ex. E.C. Hansen (Hottiger et al., 1989). Aspergillus niger van Tieghem conidiospores from a mutant deficient in mannitol production were heat-sensitive (Ruijter et al., 2003). A protective role was confirmed when exogenous mannitol restored thermotolerance in the mutants. Evidence for protein stabilization by CC has been amply demonstrated, but the mode of action is subject to several interpretations (Ganea and Harding, 2005). This is not surprising because protein unfolding by chemical denaturants is also an incompletely understood phenomenon (Bennion and Daggett, 2004). Although heat shock proteins and CC can protect proteins from denaturation, the mechanisms differ. Some families of heat shock proteins bind to exposed hydrophobic patches, preventing aggregation or misfolding of partially unfolded proteins (‘‘molecular chaperone’’ activity) (Jacob et al., 1995). Chemical chaperones appear to maintain native protein structure through interactions with the protein and solvent but may have some benefit in reducing aggregation of unfolded proteins. Although the precise nature of the interaction among protein, CC, and solvent is not clear, it is likely to be affected by chemical properties of each (Kaushik and Bhat, 2003). One way to address this dilemma is to use a complex mixture of proteins that represents a range in protein physical properties to assess CC action (Owusu and Cowan, 1989). Although information on CC activity in complex mixtures is lacking, a number of researchers have explored stabilization of individual proteins. Within limits, protein stability increased with increasing concentration (Back et al., 1979) and size (Davis-Searles et al., 2001) of polyol. Although significant progress has been made in modeling protein–solvent–CC interactions, this remains a controversial area. One approach is to view CC as a subset of cosolvents that exhibit a continuum of colligative and noncolligative effects on protein structure and function (Davis-Searles et al., 2001). Cosolvents can be considered destabilizing or stabilizing depending, in part, on the relative affinity of protein for the cosolvent and water. Attempts to model and quantify CC action include both interactions at the protein surface (preferential solvation) and changes in free energy associated with exposing the hydrophobic core on unfolding. If the driving forces for protein–solvent–cosolvent interactions are a composite of the changes in free energy associated with replacing individual solvent molecules with cosolvent molecules, mixtures of different cosolvents should have additive effects on protein stability. Received for publication 10 July 2006. Accepted for publication 28 Sept. 2006. Approved for publication by the Director, Okla. Agr. Exp. Stn. Research supported by the Okla. Agr. Exp. Stn. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product and does not imply its approval to the exclusion of other products or vendors that also may be suitable. Use of the Controlled Environment Research Laboratory and technical assistance from Shakuntala Fathepure are gratefully acknowledged. Correspondence. E-mail: [email protected] J. AMER. SOC. HORT. SCI. 132(1):67–72. 2007. 67 JOBNAME: jashs 132#1 2007 PAGE: 1 OUTPUT: January 10 14:50:06 2007 tsp/jashs/133030/00915