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Thermal Stability of Fraction I Protein from Heat-Hardened Phaseolus acutifolius Gray, ‘Tepary Buff’¹

Heat hardened ‘Tepary Buff’ bean plants were shown to have significantly greater heat tolerance than unhardened plants using a modified electrical conductivity 1nethod for assessing the heat injury. The objectives of the investigation were to determine whether heat hardening increases the thermal stability of a major leaf protein, and whether protein disulfide bonds contribute to the thermal stability. Fraction I protein was isolated from the plants by fractional ammonium sulfate precipitation and filtration through Sephadex. The thermal stability of the isolated protein was tested by changes in ultraviolet light absorption and scattering as the protein denatured at elevated temperatures. Fraction I extracted from heat hardened plants was found to be more heat stable than that from unhardened plants. Blocking the sulfhydryl groups with para-chloromercuribenzoate did not change the relatively greater thermal stability of protein from the hardened plants, whereas cleavage of disulfide bonds with mercaptoethanol and sodium sulfite decreased the stability of protein from the hardened plants and caused them to respond to increased temperatures much the same as protein from the unhardened plants. Disulfide bonds were measured quantitatively, after the sulfhydryl groups were blocked at the time of homogenizing the leaves, by the method of argentometric amperometric titration after reduction with mercaptoethanol or sodium sulfi(e. There was no significant difference found between the number of disulfide bonds in the hardened and unhardened plants. It is suggested that there may be a disulfide interchange during hardening which results in a repositioning of the disulfide bonds in such a way as to increase the thermal stability of the fraction I protein.

Key Words: disulfide bonds • bean plants

² Assistant and Associate Professors, Department of Horticulture and Forestry, University of Nebraska, Lincoln, Nebr.

Received for publication November 12, 1966.