Abstract
Previous studies had indicated that recombinant and natural human sickle hemoglobin had similar chemical properties (Martin de Llano, J. J., Schneewind, O., Stetler, G., and Manning, J. M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 918-922). In the present study, additional biochemical and physiological characterization of some primary through quaternary structural features of recombinant sickle hemoglobin are described. The molecular weight of the purified recombinant sickle hemoglobin was identical to natural sickle hemoglobin as determined by mass spectrometry, thus excluding extensive post-translational modification in the yeast system. Carboxypeptidases A and B together catalyzed the release of COOH-terminal amino acids at the same rate for recombinant and natural hemoglobin S, consistent with identity in their primary and secondary structures in this region of the molecule. The tryptic peptide maps of natural and recombinant hemoglobins were practically indistinguishable, indicating the same internal protein sequences for recombinant and natural hemoglobins. As a probe of the secondary structure of 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 position of glutathione at Cys-93(beta) was established by direct mass spectrometric analysis of enzyme digests; reduction of this derivative to the unmodified chains was also observed by mass spectrometry and by isoelectric focusing. The oxygen equilibrium curves of recombinant and natural sickle hemoglobin at high protein concentration were superimposable with identical Hill coefficients of 3.3. The response of recombinant sickle hemoglobin to chloride with respect to a lowered oxygen affinity was identical to that of natural sickle hemoglobin. The gelation properties of recombinant and natural sickle hemoglobins were identical at the high hemoglobin concentrations that occur in the red cell. Therefore, the yeast expression system synthesizes a completely functional recombinant sickle hemoglobin with the same biochemical and physiological properties as natural sickle hemoglobin with respect to features characteristic of its primary through quaternary structures.
Highlights
Griffin Rodgers From the Molecular Hematology Unit, Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Marylan2d0892
The yeast expression systemsynthesizes a completely functional recombinant sickle hemoerties that were evaluated, we found that the human sickle hemoglobin produced in yeastbehaved likenatural hemoglobin S [9]
80%of the heme-containing protein was retainbeyd the resin as deter- sium phosphate, pH 6.0, the treated natural hemoglobin was purified mined by the absorbance in the 530-580 nm range for the initial and on a Toyo-Pearl CM 650 column (25 x 10 cm),which was eluted witha final filtrates
Summary
Isolation ofNatural Sickle Hemoglobin-It was consideredimportant to document the source of the natural sickle hemoglobin samples that were used for comparison with the recombinant sihcekmleoglobin. Preparation of Glutathione Derivative of Hemoglobins-Natural or Another 0.6 ml of the resin was added to the filtrate and the mixture recombinant HbS wasincubatedwith 200 m~ oxidized glutathione was filtered again as describedabove. 80%of the heme-containing protein was retainbeyd the resin as deter- sium phosphate, pH 6.0, the treated natural hemoglobin was purified mined by the absorbance in the 530-580 nm range for the initial and on a Toyo-Pearl CM 650 column (25 x 10 cm),which was eluted witha final filtrates. The absorbance treated with a varietyof reducing agents including 2-mercaptoethanol, of the elute was monitoreadt 280 and 540 nm Those fractions contain-dithiothreitol, or glutathione for varying times a t different concentraing hemoglobin were pooled, bubbled with CO, and kept at-80 "C for tions (1-50 m)a t room temperature.
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