Hemin (Fe3+)− and Heme (Fe2+)−Smectite Conjugates as a Model of Hemoprotein Based on Spectrophotometry

Hemin (Fe3+) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin−smectite conjugate. The hemin−smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as ε398nm = 11.6 × 104 M-1 cm-1, which is in good agreement with ε398nm = (12.2 ± 3) × 104 M-1 cm-1 of monomeric hematin ( ). Hemin (Fe3+)−smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe3+)−smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe2+)−smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of α, β, and Soret bands were located in only a 9−3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe2+)−smectite conjugate was bound to O2 to form O2−heme (Fe2+)−smectite conjugate. The addition of carbon monoxide, CO, to O2−heme (Fe2+)−smectite conjugate caused the formation of CO−heme (Fe2+)−smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8−10 nm to shorter wavelength. Therefore, the transformation of O2−heme (Fe2+)−smectite conjugate to CO−heme (Fe2+)−smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the α, β, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O2−heme (Fe2+)−smectite conjugate and CO−heme (Fe2+)−smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe3+) or heme (Fe2+), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.