Determination of the amounts and oxidation states of hemoglobins M Boston and M Saskatoon in single erythrocytes by infrared microspectroscopy.

The reduced abnormal subunits of two M-type hemoglobins, Boston (His alpha 58-->Tyr) and Saskatoon (His beta 63-->Tyr), have been determined in the presence of normal human hemoglobin A by measurement of C-O stretch bands in infrared spectra of carbon monoxide complexes. Use of an infrared microscope coupled to a Fourier transform infrared spectrometer of high sensitivity permitted measurements to be made on as small a hemoglobin mixture as is contained in a single erythrocyte. The abnormal subunits of both Hbs M exhibit bands near 1970 cm-1 compared with bands near 1951 cm-1 for the normal subunits. The increase in 1970 cm-1 band intensity upon erythrocyte reduction with dithionite provided a measure of the extent of abnormal subunit oxidation; in cell suspensions about 60% of the abnormal subunits of Hb M Boston and 80% for Hb M Saskatoon remained reduced. The amount of Hb present as abnormal Hb averaged about 25% for Hb M Boston cells and about 50% for Hb M Saskatoon cells. However, the ratio of Hb M to Hb A in individual cells varied markedly, with the ratio expected to decrease as the cell ages. These results demonstrate the unique utility of infrared microspectroscopy for the study of differences in abnormal Hb status among individual erythrocytes.