Hemin control of globin synthesis: An assay for the inhibitor formed in the absence of hemin and some characteristics of its formation

Abstract

In the absence of hemin, an inhibitor of globin chain initiation is formed in the ribosome-free supernatant fraction of rabbit reticulocytes. The inhibitor may be assayed in a hemoglobin-synthesizing system containing the complete lysate and hemin. The potency of a unit of inhibitor is not altered over a broad hemin concentration range. A lag phase in inhibitor formation of two and four hours occurs if the supernatant fraction is incubated at 25 °C and 20 °C respectively. Although hemin almost completely suppresses formation of inhibitor if present from the beginning of an incubation, it only partially suppresses formation if added at the end of the lag phase. This is interpreted as indicating the presence of an intermediate in the conversion of a pro-inhibitor to inhibitor. Hemin blocks the formation of the intermediate but not its conversion to inhibitor. The formation of inhibitor continues for eight to twelve hours at 34 °C but stops in one hour at 50 °C, suggesting the possible participation of a heat labile factor. This is supported by the observation that a mixture of two supernatant fractions, one blocked in inhibitor formation with hemin and the other by heating at 50 °C, can form additional inhibitor at 37 °C.