Heat Stability of Pepper Leaf Extracts

Abstract

Acute high-temperature stress affects plant protein structure, leading to denaturation and aggregation. Although folding states of individual proteins have been extensively studied, little information is available on protein thermostability in complex mixtures. The objective of this study was to systematically examine protein stabilizing and destabilizing factors in pepper (Capsicum annuum L.) leaf extracts using light transmission measurements. Increasing turbidity and subsequent precipitation were monitored in heated extracts as changes in light scattering at 540 nm. Factors evaluated included leaf tissue concentration, buffer pH, compounds that can stabilize enzymatic activity (chelating agent, complexer of phenolics, and a compatible solute), and destabilizing agents (nonionic detergent and divalent cation). Leaf extract thermostability decreased with increasing tissue concentration from 6 to 60 g fresh weight per liter of buffer. Turbidity and precipitation occurred after exposure to higher temperatures as buffer pH increased from 6.0 to 7.0. Ethylenediaminetetraacetic acid (chelating agent) and polyvinylpolypyrrolidone (complexer of alkaloids and phenolics) had relatively small effects on extract thermostability. Nonionic detergent (Tween 20) destabilized extract thermostability, especially when incorporated in the extraction buffer. Calcium reduced thermostability by about 2 °C when added as CaCl2 at 1 mm. Calcium caused an increase in turbidity that was not directly associated with protein complexes and was not affected by treatment temperature. Mannitol, a compatible solute, increased the temperature at which turbidity and precipitation were induced, but only at high (500 mm) concentrations. Agents that stabilize or destabilize proteins at high temperatures can be assayed in plant extracts by measuring turbidity changes at 540 nm. These findings can be applied to functional studies determining the basis for differences in thermotolerance between genotypes and between control and acclimated tissues.