Studies On Biochemical Analysis With Hemin

Studies on heme and heme-containing protein are hot topics due to their broad application in biology, medicine and environmental science, this paper has described the studies on biochemical analysis with hemin based on the recent advance of heme.The determinations of trace hydrogen peroxide and glucose in serum are valuable in clinic biochemistry. Sepharose 4B, agarose, cellulose microcrystalline and permutite was respectively used as the vehicle to immobilize hemin to make the columns, which were applied to measure trace hydrogen peroxide. Furthermore, glucose concentration in serum was measured by the method of glucose oxidase(GOD) conjugating mimic enzyme, and the measurement sensitivity of these columns were examined. Results showed that hemin may be immobilized well by these activated vehicles. The hemin columns presented a stable activity of hemin and a high measurement sensitivity, but the measurement sensitivity of hemin was different in different vehicles as following: Sepharose 4B> agarose > cellulose microcrystalline > permutite. Manufacture of hemin column was cheap, the column may be used repeatedly and applied for quick examination of trace hydrogen peroxide or glucose concentration in serum.Equimolar BSA and hemin were combined in vitro to form hemalbumin based on the vivid property of bovine serum albumin(BSA), of which the peroxidase activity was examined . Results showed that hemalbumin possessed the peroxidase property, and it was a stable, active molecule. Then hemalbumin was immobilized to the activated vehicle Sepharose 4B with the couple ratio of 5.3mg hemalbumin per gram vehicle to make hemalbumin column, which was applied to measure trace hydrogen peroxide. Hemalbumin column presented a high measurement sensitivity, a stable peroxide as low as 3.63×10^-7mol/L. This experiment went step further to explore that synthesized hemalbumin was used in the catalytic oxidation of p-cresol by hydrogen peroxide. The peroxidase characteristics of hemalbumin and enzymatic kinetics were studied. The K_m value(1.85×10^-3mol/L) and V_m value(1.6min^-1) were measured by steady-state catalytic velocity at pH=10.3 and temperature 25℃, The reaction mechanism was discussed when p-cresol was used as a hydrogen donor for hemalbumin. The velocity equation...