Analyzing bean extracts using time-dependent SDS trapping to quantify the kinetic stability of phaseolin proteins.

ABSTRACT

In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60-75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20-100 years at 24 °C and 2-7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification.