TCA and washed three times with 1.0 ml of an ethano1/ethyl acetate
mixture (1:l). The ethano1/ethyl acetate extract was virtually
colourless by the third wash, indicating complete removal of
unreacted DNPH. The pellet was dried and dissolved in 2 ml of
6 M guanidine dissolved in 20 mM potassium phosphate (pH
2.3). The protein concentration was determined using a Bio-Rad
Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) based
on the Bradford method. The difference in absorbance at 370 nm
between HC1-treated samples and DNPH-treated samples was
taken as a measure of reacted carbonyl groups using a molar
extinction coefficient of 2.2 104 M1 cm1.