The binding of pyridoxal to hemoglobin.

Concentrative uptake of pyridoxal by human erythrocytes has been investigated using a rapid mixing technique with 3H-labeled pyridoxal. A nonsaturable, initial influx of [3H]pyridoxal into the erythrocyte indicated passive diffusion. Since pyridoxal will form Schiff bases reversibly with amino acids, the possibility of binding to intracellular proteins was examined. Exposure of erythrocytes to pyridoxal followed by NaBH4 reduction, resulted in a stable pyridoxyl-protein complex. The binding site for pyridoxal was found to be on the alpha chain of oxyhemoglobin, as determined by ion exchange chromatography and amino acid analysis. Separation of the tryptic peptides from the [3H]pyridoxyl-alpha chain on Dowex 50 and analysis of the [3H] pyridoxal peptide showed that the binding site of pyridoxal was the NH2-terminal valine. Pyridoxal was also found to bind to the alpha chain of nonoxygenated hemoglobin. The rate of pyridoxyl-hemoglobin formation in a cell-free system provided additional evidence in support of the suggestion that concentrative uptake of pyridoxal by human erythrocytes is due to intracellular binding of pyridoxal to hemoglobin. Pyridoxal phosphate and glucose, which also bind to the NH2-terminal valine, did not reduce the accumulation of pyridoxal in the erythrocyte.