Identification and analysis of a non-major band of human hemoglobin in SDS-PAGE

To characterize the non-major band of human hemoglobin (Hb) detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Adult Hb was purified from human whole blood via erythrocyte lysis combined with chromatography. The purity and band distribution of the purified products were analyzed using coomassie brilliant blue staining and Western blotting. Samples were treated with iodoacetamide (IAM), sodium dodecyl sulfate (SDS), and dithiothreitol (DTT) to identify the dimeric forms and their distribution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to determine the molecular masses while ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed to analyze the protein composition and relative abundance.

Western blotting revealed a major monomer band (band 1) at a relative molecular weight of 14-15×10³ Da and a specific non-major band (band 2) between 25 and 35×10³ Da. Relative quantitative analysis showed that hemoglobin subunit beta (HBB) was significantly higher than hemoglobin subunit alpha (HBA) in band 2 and the band 2/band 1 ratio. After enhanced-reduction treatment, the signals decreased for HBA in band 2, and for HBB in band 1 and band 2 as well, while the band 2/band 1 ratio increased. MALDI-TOF MS confirmed that the purified Hb was mainly composed of HBA, HBB, and heme. UHPLC-MS/MS demonstrated that both band 1 and band 2 were predominantly HBA and HBB, with small amounts of hemoglobin subunit delta (HBD), hemoglobin subunit gamma 1 (HBG1), and hemoglobin subunit gamma 2 (HBG2). HBB was more abundant than HBA in both bands, with a higher HBB/HBA ratio in band 2 than in band 1.

The non-major band of human Hb is mainly composed of HBA and HBB. The proportion of HBB is higher than that of HBA in the major band, which further increases in the non-major band, reflecting differences in molecular stability and aggregation kinetics between HBB and HBA.