Study On Purification Of Heme-binding Protein From Escherichia Coli Using Hemin Affinity Chromatography

Heme-binding protein (HBP) generally belongs to globulin, in which there is at least one or several heme binding domain that can bind with heme. So far HBP is considered to have many physiological functions, including oxygen sensor, free heme transfer, participating in metabolic and so on. But there still are many to be probe for its functions and work mechanism. How the protein molecules to utilize the heme to perform different biological function, is always the research focus in chemical biology and protein chemistry. The researchers at home and abroad proceeded on it long and extensive, and tried to understand this question to recognize the relationship among structure-property-reactivity-function (SPRF) of proteins. Obviously, study on HBP is helpful for probing this question. So in this study, based on the mechanism of affinity chromatography, the affinity chromatography column with the heme immobilized was built to do purification of HBP.After inertia molecular sieve Sephadex G25 polymer beads were activated by using epichiorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column. Spectral analysis showed that the coupling rate of this column was very high. More than 1.56 mg hemin could be fixed by 1g of wet Sephadex G25 beads. The affinity chromatography column was very specific and efficient for extraction and purification of HBP. Two kinds of polypeptides were captured by using this column from Escherichia coli, one strap was about 33 KDa, and another was about 15 KDa demonstrated by SDS-PAGE. Vis-UV spectral analysis explained that these polypeptides had interaced with hemin to possess the properties of HBP. Using this affinity chromatography system can purify HBP quickly and specifically.