A rapid method for hemoglobin chain recombination.
A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of alpha- and beta-chains were mixed with 300 x molar excess of beta-mercaptoethanol over the p-hydroxymercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mM phosphate buffer-1 mM Na2EDTA-47 mM beta-mercaptoethanol, pH 5.85 and then with 10 mM phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mM K2HPO4. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.
Lee
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Center for Sickle Cell Disease, Howard University College of Medicine, Washington, D. C. 20059, USA.
D002266:Carboxymethylcellulose Sodium; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D006902:Hydroxymercuribenzoates; D007202:Indicators and Reagents; D008623:Mercaptoethanol; D011487:Protein Conformation
D006454:Hemoglobins; D006902:Hydroxymercuribenzoates; D007202:Indicators and Reagents; C030601:4-hydroxymercuribenzoate; D008623:Mercaptoethanol; D002266:Carboxymethylcellulose Sodium