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J. Biochem, 1984, Vol. 96, No. 4 985-991
© 1984 Japanese Biochemical Society

research-article

Further Improvement in Preparation and Some Properties of Native {alpha} and ß Chains from Canine Hemoglobin Treated with p-Chloromercuribenzoate

Susumu TOMITA1, Susumu SAKATA, Yasunori ENOKI and Hisaharu KOHZUKI

Department of Physiology, Nara Medical College Kashihara, Nara 634

1To whom all correspondence should be addressed.

The {alpha} and ß chains were prepared from canine hemoglobin by a modification of the method of Bucci and Fronticelli ((1965) J. Biol. Chem. 240, PC551–552) which involved treatment of hemoglobin with an excess of p-chloromercuribenzoate (pCMB), separation of the mercurated chains by chromatography on a DE-32 column65 with a salt gradient at pH 8.6, and regeneration of sulfhydryl groups of the chains with 2-mercaptoethanol. The SH titer was two per heme for both the regenerated {alpha} and ß chains. The titer decreased to four per tetramer of hemoglobin after equimolar recombination of both chains. Measurements of the absorption spectrum and oxygen binding showed that the chains were in a native state.


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