His64(E7)–>Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.

Abstract

To develop an assay for hemin dissociation, Hiss4(E7) was replaced by % in sperm whale myoglobin producing a holoprotein with a distinct green color due to an intense absorption band at 600 nm.Valee(E1l) was replaced by Phe in the same protein to increase its stability.When excess w -V a l s * apoglobin is mixed with either metmyoglobin or methemoglobin, the solution turns from brown to green, and the absorbance changes can be used to measure complete time courses for hemin dissociation from either holoprotein.This assay has been used to measure rates of hemin dissociation from native metmyoglobin, four myoglobin mutants (Alas4(E7), Alaee( E1l), Phese(Ell), and G ~U ~~( C D ~) ) , native methemoglobin, valence hybrid hemoglobins, and two mutant hemoglobins ((a(Gly-E7)P(native))~, and ((~(native)P(Gly-El))~).Two kinetic phases were observed for hemin dissociation from native human hemoglobin at pH 7.0 and 37 "C.Valence and mutant hybrid hemoglobins were used to assign the faster phase (k = 7.8 t 2.0 h-l) to hemin dissociation from ferric P subunits and the slower (k = 0.6 0.16 h-l) to dissociation from (Y subunits.The corresponding rate for wild-type metmyoglobin is 0.007 t 0.004 h-l.A general scheme for hemoglobin and myoglobin denaturation is shown below (adapted from Winterbourn and Carrel1 (1977) and Bunn and Forget (1986)). m o , tA ZHb'(H,O) a hemin + globin-precipitates (red cell lysis) / 2H' + 20;-0, + H,O, irreversible hemichromes (Eq. 1)