Lee K S, Huang P C, Cohen B H
Biochim Biophys Acta. 1976 Mar 18;427(1):178-96. doi: 10.1016/0005-2795(76)90295-6.
Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.
有证据表明，成年鸡的血红蛋白存在四种类型，通过在聚丙烯酰胺凝胶上进行等电聚焦可将其分离，而不是像之前用其他方法分离出的两种血红蛋白类型。这四种类型分别是血红蛋白A1（HbA1）、血红蛋白A2（HbA2）、血红蛋白D1（HbD1）和血红蛋白D2（HbD2）。它们的等电点（pI）值分别为7.53±0.02、7.37±0.02、6.92±0.04和6.72±0.05，分别约占成年鸡红细胞总血红蛋白的63%、14%、18%和5%。在十二烷基硫酸钠凝胶上测定发现，HbA1和HbA2大小相同，它们的氨基酸组成和胰蛋白酶肽也相似。尽管HbD1和HbD2的胰蛋白酶肽存在差异，但它们的分子量和氨基酸组成相同。实验表明，四种血红蛋白类型的存在并非由于实验动物的遗传异质性，也不是由于羧基血红蛋白氧化为高铁血红蛋白四聚体的假象。通过使用新鲜制备的溶血产物来消除侧链氨基的脱氨和修饰，并通过控制电泳时间将等电聚焦特有的“平台现象”降至最低。本研究中使用氰化高铁形式（即配体化）的血红蛋白减少了异源四聚体形成的可能性。此外，还考虑了两性电解质可能引起的异常情况。鉴于没有假象的负面证据，得出的结论是成年鸡的血红蛋白类型不止之前报道的两种。
