BIOCHEMICAL AND FUNCTIONAL-PROPERTIES OF RECOMBINANT HUMAN SICKLE HEMOGLOBIN EXPRESSED IN YEAST

Previous studies had indicated that recombinant and natural human sick le hemoglobin had similar chemical properties (Martin de Llano, J. J., Schneewind, O., Stetler, G., and Manning, J. M. (1993) Proc. Natl. Ac ad. Sci. U. S. A. 90, 918-922). In the present study, additional bioch emical and physiological characterization of some primary through quat ernary structural features of recombinant sickle hemoglobin are descri bed. The molecular weight of the purified recombinant sickle hemoglobi n was identical to natural sickle hemoglobin as determined by mass spe ctrometry, thus excluding extensive post-translational modification in the yeast system. Carboxypeptidases A and B together catalyzed the re lease of COOH-terminal amino acids at the same rate for recombinant an d natural hemoglobin S, consistent with identity in their primary and secondary structures in this region of the molecule. The tryptic pepti de maps of natural and recombinant hemoglobins were practically indist inguishable, indicating the same internal protein sequences for recomb inant and natural hemoglobins. As a probe of the secondary structure o f recombinant sickle Hb, the reactivity of the SH group of Cys-93(beta ) was investigated for the glutathione sickle hemoglobin adduct, which has significant anti-gelling and anti-sickling properties. The positi on of glutathione at Cys-93(beta) was established by direct mass spect rometric analysis of enzyme digests; reduction of this derivative to t he unmodified chains was also observed by mass spectrometry and by iso electric focusing. The oxygen equilibrium curves of recombinant and na tural sickle hemoglobin at high protein concentration were superimposa ble with identical Hill coefficients of 3.3. The response of recombina nt sickle hemoglobin to chloride with respect to a lowered oxygen affi nity was identical to that of natural sickle hemoglobin. The gelation properties of recombinant and natural sickle hemoglobins were identica l at the high hemoglobin concentrations that occur in the red cell. Th erefore, the yeast expression system synthesizes a completely function al recombinant sickle hemoglobin with the same biochemical and physiol ogical properties as natural sickle hemoglobin with respect to feature s characteristic of its primary through quaternary structures.