Abstract
It has been known that organic phosphate compounds, such as 2, 3-diphosphoglycerate and ATP, interact with hemoglobin molecule to lower its oxygen affinity and to increase the Bohr effect. There are many reports which are concerned with the interaction of 2, 3-diphosphoglycerate and human hemoglobin. However, it is not always feasible to identify amino acid residue (s) in the hemoglobin molecule involved in the interaction which is electrostatic in nature.
The author attempted an experiment to clarify binding properties between pyridoxal 5′-phosphate which is bifunctional in the interaction with proteins and the two hemoglobins from eel, Anguilla japonica, differing from one another in their structure and function.
Results obtained are as follows:
1) Pyridoxal 5′-phosphate considerably lowers the oxygen affinity of E1 hemoglobin, while it scarcely affects that of E2 hemoglobin.
2) In the presence of both pyridoxal 5′-phosphate and inositol hexaphosphate, the oxygen affinity of E1 hemoglobin is higher than that of the former alone and lower than that of the latter alone, respectively, indicating that these phosphate compounds compete for the same site (s) on the E1 hemoglobin.
3) The stoichiometry of the covalent binding reaction of pyridoxal 5′-phosphate and hemoglobin is two moles of pyridoxal 5′-phosphate per mole of E1 deoxyhemoglobin with a binding constant of 1.4-104M-1, and one mole per mole of E2 deoxyhemoglobin with 2.4-103M-1, respectively.
4) Two moles of pyridoxal 5′-phosphate bound with hemoglobin are found in one of the two constituent subunits (the α and β chains). It is most conceivable that N-terminal amino group of the β chain which is not acetylated binds with aldehyde group of pyridoxal 5′-phosphate to form Schiff's base.
5) E1 hemoglobin bound with pyridoxal 5′-phosphate through covalent bond showed an irreversibly lowered oxygen affinity. The maximal change in the oxygen affinity of E1 hemoglobin is observed as one mole of pyridoxal 5′-phosphate binds with the E1 hemoglobin molecule, suggesting a free amino group in either one of the two β chains, probably N-terminal amino group, as a possible binding site involved in lowering the oxygen affinity of E1 hemoglobin.
