Recombinant sickle hemoglobin containing a lysine substitution at Asp-
85(alpha): expression in yeast, functional properties, and participation in
gel formation
JP Himanen, AM Popowicz and JM Manning
The Rockefeller University, New York, NY, USA.
Clinical modalities based on inhibition of gelation of HbS are hindered by
the lack of quantitative information on the extent of participation of
different amino acid residues in the aggregation process. One such site is
Asp-85(alpha), which is involved in a parallel interdouble strand ionic
interaction with Lys-144(beta) according to the crystal structure of HbS,
but electron microscopy does not specifically show Asp-85(alpha) as a
contact site for fiber formation. Using a yeast recombinant system, we have
substituted this site by Lys to abolish ion pairing and to make a
quantitative determination of its participation in aggregation. The
purified double mutant was shown to have the expected pI, the calculated
molecular weight, correct amino acid composition, and peptide map. The
recombinant double mutant has an oxygen affinity of 10 mm Hg, which is
identical to that for HbA and HbS under the same conditions; it also has
high cooperativity with an average n value of 2.7. The change in P50 in
response to chloride ions was about 25% less than that for HbA or HbS and
is ascribed to the introduction of a new positive charge near one of the
major oxygen- linked chloride binding sites of hemoglobin. The gelation
concentration of the double mutant was measured by a new procedure
(Bookchin et al, 1994); the maximal amount of soluble hemoglobin (Csat) in
the presence of dextran indicated a decreased tendency for gelation with a
Csat of 53 mg/mL compared with 34 mg/mL for HbS. This inhibitory effect is
smaller than that of the E6V(beta)/L88A(beta) (Csat, 67 mg/mL) and the
E6V(beta)/K95I(beta) (Csat, 90 mg/mL) recombinant hemoglobins. Thus, we
would classify Asp-85(alpha) as a moderate contributor to the strength of
the HbS aggregate. This wide range of gelation values demonstrates that
some sites are more important than others in promoting HbS aggregation.
Volume 89,
Issue 11,
pp. 4196-4203,
06/01/1997
Copyright © 1997 by The American Society of Hematology
