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(a lpha 58) --> Tyr) and Saskatoon (His(beta 63) --> Tyr), have been dete rmined in the presence of normal human hemoglobin A by measurement of C-O stretch bands in infrared spectra of carbon monoxide complexes. Us e of an infrared microscope coupled to a Fourier transform infrared sp ectrometer of high sensitivity permitted measurements to be made on as small a hemoglobin mixture as is contained in a single erythrocyte. T he abnormal subunits of both Hbs M exhibit bands near 1970 cm(-1) comp ared with bands near 1951 cm(-1) for the normal subunits. The increase in 1970 cm(-1) band intensity upon erythrocyte reduction with dithion ite provided a measure of the extent of abnormal subunit oxidation; in cell suspensions about 60% of the abnormal subunits of Hb M Boston an d 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% fo r Hb M Saskatoon cells. However, the ratio of Hb M to Hb A in individu al cells varied markedly, with the ratio expected to decrease as the c ell ages. These results demonstrate the unique utility of infrared mic rospectroscopy for the study of differences in abnormal Hb status amon g individual erythrocytes.