In 1968 Schroeder et al.' described a heterogeneity of human fetal haemoglobin resulting from the presence of either a glycyl (Gy) or an alanyl residue (Ay) in position 136 of the y-chain. In 1976, Ricco et al.* discovered another type of y-chain heterogeneity in which the isoleucyl residue in position y 75 was replaced by a threonyl. Ay and Gy chains are products of two non-allelic structural genes, while AyI and AyT chains are produced by alleles of the Ay chain gene.
The determination of the y-chain heterogeneity of haemoglobin F is of interest for genetic and population studies 3. It may also yield some information about the biological and clinical polymorphism of haemoglobinopathies4.
The first determinations of the Gy and Ay chain ratio were conducted by various chromatogrpahic procedures followed by amino acid analyses. Improvement in the methodology, brought by high-performance liquid chromatographic (HPLC) and by electrophoretic techniques have allowed extensive studies. Congote et al.5 and Shelton et ~1.~ described the separation of the a, /?, Gy and AyI globin chains by HPLC on a Cl8 100-A porosity column packing but with different solvent systems. Huisman et al.' made some modifications to the system of Shelton et uL6 so that the three types of y-chains may be separated in 3 h. More recently a perchlorate phosphate-methanol-acetonitrile system was proposed by Shelton et ~1.~ which, in 80 min, allowed a qualitative detection of the AyT chain.
In the present communication we describe a modification of the HPLC procedure using a 300-A porosity column packing and trifluoroacetic acid (TFA)acetonitrilemethanol as eluent. This procedure allows, in 80 min, with a reduced flow-rate as compared to the previously described methods, the quantitation of the three types of y-chains. In addition, the completely volatile elution system described may be of interest for preparing salt-free material for further investigations.
Blood was drawn in heparinized tubes and washed red blood cells were lyzed with water. Haemoglobin solutions were purified with toluene and stroma removed by centrifugation (13,000 g; 5 min). Globin was prepared by the acid-acetone method. For each sample, 5-10 mg of globin were prepared. Before analysis the freeze-dried globin was dissolved in distilled water at a concentration of cu. 5 mg/lOO ~1.
An Aquapore RP 300 column (25 x 0.46 cm) (Brownlee Labs., Sante Clara, CA, U.S.A.) protected by a guard column was used. With a flow-rate of 1 ml/min,