Chemical modification of heme group improves hemoglobin affinity for hydrophobic substrates in organic media

Carboxylic groups of heme prosthetic group from hemoglobin were modified with p-nitrophenol and p-aminophenol, using a carbodiimide chemistry, to change its electron character and to increase its hydrophobicity. The modification of heme group included the extraction of heme group by the method of acid acetone, the chemical modification of protoporphyrin IX and the reincorporation of modified heme group in apohemoglobin. The effect of the chemical modification on substrate affinity and catalytic activity were studied. Dissociation constants in aqueous media using different substrates showed that chemical modification of hemoglobin active-site improved the substrate affinity up to 30 times. In addition, the chemical modification slightly increased the solvent concentration at which hemoglobin was catalytically active. This biocatalytic behavior could be attributed to the hydrophobicity increase of active site. On the other hand, the chemical modification of the heme prosthetic group altered its electron balance affecting the specific activity of hemoglobin.