CHANGES OF THE γ ISOFORM COMPOSITIONS IN FETAL HEMOGLOBIN DURING POSTNATAL DEVELOPMENT

An efficient procedure was developed for the enrichment of Hb F (α₂ γ₂) in human adult hemolysate by an alkali denaturation-salting out. The method can be applied to small amounts of blood with less than 1% of Hb F, and provides a sufficient Hb F level to allow an accurate high-performance liquid chromatographic (HPLC) quantitation of the three γ chain isoforms, ᴳγ (136 Gly, 75 Ile), ᴬγᴵ (136 Ala, 75 Ile) and ᴬγᵀ (136 Ala, 75 Thr). CM-Sephadex chromatography can be combined to attain a further enrichment. Hb F isolated from adult blood was essentially identical with the protein from cord blood in their physicochemical and physiological properties. Developmental changes in the γ isoform composition of Hb F were studied in 110 normal Japanese newborns, 297 infants within one year after birth and 121 adults, first by the γCB-3 peptide procedure and then by HPLC method. The average ratio of ᴳγ/ᴬγ changed from 7/3 at birth to 4/6 during the postnatal 4- to 5-month period, and thereafter settled down gradually to 3/7 in the adults. The most conspicuous finding was that the isoform composition and therefore the γ gene expression, which was kept within a strictly confined level in the neonatal period, was rendered gradually to show a highly extensive variation during postnatal development. The ᴬγᵀ gene frequencies in the newborns and the adults were estimated as 0.125 and 0.160, respectively, and the values were consistent with those previously reported for Japanese subjects, but quite different from those for Chinese and Afro-americans. The γ isoform compositions were also studied in multiple pregnancies. The results suggested the presence of an "external" factor affecting the expression of the γ globin genes and also the possibility of using the γ isoform compositions to determine the zygosity of twins, albeit under limited conditions