The effects of temperature and pH on secondary structure and antioxidant activity of Crocodylus siamensis hemoglobin.

Crocodylus siamensis hemoglobin (cHb) was purified by gel filtration chromatography and visualized by SDS-PAGE. Effects of temperature and pH on secondary structure and conformation changes of cHb were studied using circular dichroism spectropolarimeter and fourier transform infrared spectrophotometer. The secondary structure of intact cHb was mainly α-helices. cHb was not heat stable when heated at 65 °C and cooled down to original temperature, indicating the irreversible unfolding process. The stability of cHb at different pH ranging from 2.5 to 10.5 was determined. The maximum value of the α-helix content was found at pH 3.5 and tended to decrease at strong acid and strong base. The antioxidant activities of heat treated cHb and cHb in solution with pH range 2.5 to 10.5 were tested by DPPH radical scavenging assay. cHb at pH 4.5, having highest β-turn structure, showed highest radical scavenging activity. In contrast to pH, heat had no effect on antioxidant activity of cHb.