HEMOGLOBIN BIOSYNTHESIS IN RETICULOCYTES OF A HEMOGLOBIN M HYDE PARK PATIENT
Reticulocytes were collected from the venous blood of a Hb M Hyde Park (Hb M HP: α2Aβ2M;βM：β 92 His→Tyr) patient, and they were incubated with Lingrel-Borsook culture medium containing 3H-Leu for hemoglobin biosynthesis. Hemolysate containing whole biosynthesized hemoglobins was prepared from these reticulocytes. Hemoglobins were separated individually by cellulose acetate membrane electrophoresis of metHb type hemolysate and by DEAE-cellulose column chromatography of oxyHb type hemolysate. Globins were prepared from hemoglobins and they were divided into the α and the non-α chains by urea dissociation cellulose acetate membrane electrophoresis and urea CM-cellulose chromatography. The isolated fractions of hemoglobins and globin chains thus collected were measured for their radioactivities in a liquid scintillation counter. It was shown that both metHb M HP and oxyHb HP (oxyHb M Hyde Park with deletion of one molecule of heme from its abnormal βM chains) incorporated 3H-Leu 2.3 times as much as the relevant metHb A and oxyHb A did. The specific radioactivity ratio of globin chains (β/α) was only 0.45 : 1.00 (βM/αA) in metHb M HP, whereas it was 1.00 : 1.00 (βA/αA) in metHb A. In the case of globin chains prepared from the oxyHbs separated by urea CM-cellulose column chromatography of the hemolysate the radioactivity ratio βM/βA was 1.22 : 1.00, being approximately equal to 1:1. This is interpreted that the abnormal βM chain is produced at a rate nearly the same as or slightly higher than that of the βA chain. On the contrary, the radioactivity ratio of non-α chain/α chain ([βA+βM]/αA) was significantly smaller than unity, namely 0.45 : 1.00. This suggests that production of the α chain is enhanced over the level of the non-α chain (βA + βM), and there is an excess of the α chain over the non-α chains, resulting in formation of free α chain pool in the cytosol of reticulocytes of the Hb M HP patient. Exchange of α chain between the molecules of pre-existing Hb M HP～Hb HP and the free α chain pool is thought to be significantly large on account of spontaneous dissociation of the molecules of the abnormal unstable hemoglobins (Hb M HP～Hb HP; probably, α2Aβ2M→α2A+β2M).