Man Ib And X-type Phospholipase A2 In Protein Folding And Refolding

Mammalian secretory phospholipase A2 contains 6-8 disulfide bonds with a molecular mass of 13-16 kDa. It catalyzes the hydrolysis of phospholipids. Till now, 10 groups of mammalian secretory phospholipase A2 have been identified, among which group IB and X phospholipase A2 contain propeptides at the N-terminals. In this thesis, unfolding and refolding of human group IB and X phospholipase A2 have been studied. Human group IB phospholipase A2 and prophospholipase A2 were expressed in a form of inclusion body in Escherichia coli. Human phospholipase A2 and its proform were purified from inclusion bodies by unfolding and refolding. CD, fluorescence and HPLC were used to monitor the unfolding process induced by guanidine HCl and DTT. Our results showed that mature enzyme was more stable than the proform toward denaturats and DTT. Human group IB prophospholipase A2 was in a metastable state. It might be explained that the refolding of proform was over the mature form enzyme. Metastable conformation has an advantage for the folding pathway. Mutants with N-termial deletion of propeptide impair the function to assist folding. The disulfide bond 11-77 in human group IB phospholipase A2 locates at the surface of the protein and was firstly reduced by DTT. Human group X phospholipase A2 is highly homologous to human group IB phospholipase A2 both in amino acid sequence and crystal structure. The refolding of human group IB phospholipase A2 was unlike to human group X phospholipase A2. Intermolecular disulfide bonds were the main trouble. Low temperature, low protein...