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J. Biochem, 1968, Vol. 63, No. 2 193-198
© 1968 Japanese Biochemical Society

research-article

Amino Acid Substitution in Hemoglobin MAkita*

SUSUMU SHIBATA, TAKAOKI MIYAJI, IWAO IUCHI{dagger}, YUZO OHBA and KIYOMI YAMAMOTO

From the Department of Medicine, Clinical Pathology, the Yamaguchi University School of Medicine Ube
{dagger}Kawasaki Institute of Tumor Research Okayama

A new variant of hemoglobin M (Hb M) was discovered from a family of hereditary cyanosis living in Akita by combined use of conventional tests for Hb M, including spectroscopy of methemoglobin type hemolysate, agar gel electrophoresis (pH 7.0) and Amberlite IRC 50 chromatography. Hybridization test of the hemoglobin with canine hemoglobin disclosed its ß chain anomaly. The hemoglobin was called Hb MAkina

MetHb MAkita was separated from metHb A (methemoglobin of normal adult hemoglobin) by starch block electrophoresis (pH 7.0) of the metHb type hemolysate of the patient, and heme was removed from it to get MAkita globin ({alpha}A2 ßM2). Mixture of ß polypeptide chains (ßM+ßA) free from {alpha} chain was also obtained by chromatography of the globins (Hb MAkita+Hb A) prepared directly from the patient's hemolysate on a column of carboxymethyl cellulose with buffers containing 8 M urea.

The MAkita globin and the ß chain mixture were aminoethylated, and the protein derivatives were digested with trypsin [EC 3.4.4.4 [EC] ]. The peptide map (fingerprint) of the digest was prepared. The fingerprint thus made revealed abnormal spots related to peptides ßTp-10 and ßTp-10, 11. These were eluted, and hydrolyzed with hydrochloric acid. The amino acid analysis of the acid hydrolysate has demonstrated that, in Hb MAkita, tyrosine substitutes for histidine (ß 92) at the ninety-second residue of the ß chain. This hemoglobin is therefore expressed by the formula {alpha}2ß92Tyr2. By perusal of literatures it has become apparent that Hb MAkita is identical with Hb MHyde park which was detected from a negro patient in the United States several months earlier.

*Dedicated to Dr. Koozoo Kaziro, Professor Emeritus of the Nippon Medical School, on the occasion of his 70th birthday.


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