Reconstitution of human fetal hemoglobin from isolated alpha and gamma chains

The reconstitution of hemoglobin F from isolated alpha and gamma chains was studied. An equimolar amounts of the alpha and gamma chains were mixed and incubated in 10 mM potassium phosphate buffer, pH 7.0, at 25 degrees C. Formation of hemoglobin F in the mixture was measured by separating hemoglobins on a cation exchange HPLC. Time courses of the formation of Hb F were independent of the protein concentration and could be analyzed on an exponential process with a first-order rate constant of (2.0 +/- 0.4) x 10(-3) h-1. Under the experimental conditions the isolated gamma chain existed as tetramer dominantly. These results suggest that the overall reaction of the reconstitution of hemoglobin F is limited by the dissociation step of the self-associated gamma chain.     

Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.

New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with either dithiothreitol or beta-mercaptoethanol. In general the temporary blocking groups also afford total protection against normally inhibitory thiol blocking agents. When RS- groups were attached to rabbit muscle creatine kinase (EC 2.7.3.2), a trend toward lower residual activities with increasing bulk was observed. Treatment of the native creatine kinase with 14CH3HgC1 led to incorporation of greater than 1 equiv of CH3Hg- group per subunit. This CH3Hg- blocked enzyme was fully active, and the blocking group afforded no protection against iodoacetamide. These results suggest that CH3Hg- and the RS- groups are modifying two different sulhydryl groups on the enzyme. When papain (EC 3.4.4.10) was treated with excess methyl methanethiolsulfonate. complete and rapid inhibition was observed, and 1 equiv of CH3S- was incorporated/mol of active enzyme. Complete protection against normally inhibitory 5,5'-dithiobis(2-nitrobenzoic acid) was afforded by the temporary blocking group. When rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was titrated with methyl methanethiolsulfonate, two sulfhydryl groups per subunit were found to be modified, one much more rapidly than the other. If one extrapolates the initial slope of the titration curve, the inactivation of the enzyme would be complete after modification of a single cysteinyl residue per subunit.     

Separation of the A gamma and G gamma cyanogen bromide peptides of human fetal hemoglobin by high-pressure liquid chromatography.

The ^Gγ and ^Aγ variants of human fetal hemoglobin are defined on the basis of a single glycine/alanine substitution at position 136 of the γ chain which is located in the carboxyterminal cyanogen bromide fragment (γCB-3). This communication describes the rapid separation of these two tridecapeptides by high-pressure liquid chromatography. Application of this new technique will facilitate the study of the biosynthesis and genetics of human fetal hemoglobin.