Identification and Characterization of Hemoglobin in Thai Bangkaew Dogs using Chromatographic, Electrophoretic and Mass Spectrometric Techniques

Abstract

The present study was aimed to measure the hematocrit (Ht) and the hemoglobin (Hb) concentration of Thai Bangkaew dogs, to investigate Hb phenotypes based upon the electrophoretic pattern of the Hb and to estimate molecular weights of the Hb (tetramer) and Hb subunits using gel
filtration chromatography, SDS-PAGE and MALDI-TOF/TOF MS. The results showed that 30 dogs had a mean Ht value (± standard error of the mean) of 36.38 ± 0.77% and a mean Hb concentration (± standard error of the mean) of 13.10 ± 0.32 g/dL. In addition, the fraction obtained from gel filtration of Sephadex G-100, at pH 7.4, corresponding to the isolated Hb protein was estimated at a molecular weight of 65,956 Dalton. The Hb fraction was used for analysis by native polyacrylamide gel electrophoresis (native-PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). The Hb electophoretic mobility in native-PAGE showed that all samples had one two-banded phenotype, consisting of a low mobility major band (94%) and a minor fast band (6%). In addition, SDS-PAGE showed two distinct bands of approximate molecular weights 12,980 and 14,820 Dalton, respectively. Moreover, the MALDI-TOF/TOF MS analyzes on the purified Hb fraction exhibited two abundant mass peaks at the molecular weights of 15,194.78 and 15,946.66 Dalton and three minor peaks at the molecular weights of 32,020.53, 48,326.29 and 64,545.95 Dalton.