TY - JOUR
T1 - Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel
AU - Lee, Kang Sun
AU - Huang, P. C.
AU - Cohen, Bernice H.
N1 - Funding Information:
The authors wish to thank Mr. Per-Olof Backman for his assistance and advice in the amino acid analyses. The work was supported by the National Foundation/March of Dimes Grant R73126, CSBR300 (P.C.H.), and by Grant No. 5 PO1 CA 11489 from the National Cancer Institute (B.H.C.).
PY - 1976/3/18
Y1 - 1976/3/18
N2 - 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 are identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weights 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 or of aggregation due to overloading or formation of multiple units of hemoglobin 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.
AB - 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 are identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weights 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 or of aggregation due to overloading or formation of multiple units of hemoglobin 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.
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U2 - 10.1016/0005-2795(76)90295-6
DO - 10.1016/0005-2795(76)90295-6
M3 - Article
C2 - 1259997
AN - SCOPUS:0017256610
SN - 0005-2795
VL - 427
SP - 178
EP - 196
JO - BBA - Protein Structure
JF - BBA - Protein Structure
IS - 1
ER -