Study On Isolation Of Lectin, Cytochrome C And Myoglobin, On Interaction Among Heme-containing Proteins Using Chromatography

The S-type lectins in annelida are different in molecular structure and biochemical properties from that of common galectin and are valuable in anticancer study. Based on their specificity, Sepharose CL-6B was adopted in this study as affinity chromatography to separate the S-type lectin in annelid from the earthworm, in which EDTA-MEPBS (2 mmol/L EDTA, 4 mmol/Lβ—mercaptoethanol, 150 mmol/L NaC1, 20 mmol/L phosphate, pH7.2) was used as an equilibration solution and ammonia water (150 mmol/L, pH10.5) as an eluant. SDS-PAGE showed that the molecular mass of obtained protein in this purification procedure was about 32KD and it could be separated into fragmentswith molecular mass about 15KD. Hemagglutination experiment and fluorescence analysis demonstrated that this protein possessed the characteristics of agglutinin and combination with lactose. These explained that the target protein was one of S-type lectins in annelida. This study may offer a novel and rapid method for purifying the S-type lectins in annelida in large scale.A strong anion exchange chromatography Q Sepharose XL column and Sephadex G-75 size-exclusion chromatography were adopted to extract myoglobin (Mb) and cytochrome-c (Cyt C) from the pig heart. In determination ofa pH working range for Q Sepharose XL column, Cyt C was found to be captured on the column and Mb passed through slowly in pH 7.6-8.5. Ion exchange chromatography was performed at loading pH 7.9 and eluting pH 7.2. The contents of Mb and Cyt C in the samples obtained in initial purification process by using Q Sepharose XL column was 89.7% and 93.2%, respectively, as determined by SDS-PAGE. The pure Mb and Cyt C could be obtained by further adopting Sephadex G-75 size-exclusion chromatography. The productivity of Mb was finally 77.43 and Cyt C was 86.42 mg/kg. This study had demonstrated it was available for fast preparation of Mb and Cyt C from the pig heart by using Q Sepharose XL anion exchange chromatography. In this Study, interactions among human hemoglobin, earthworm hemoglobin, horse myoglobin and horse cytochrome C researched with affinity chromatography. It was showed that horse myoglobin and horse cytochrome C earthworm hemoglobin would bind to earthworm hemoglobin; and would be band to human hemoglobin too. It suggested that they have similar structure in detail in some degree and they were homologous proteins.