Conformational studies on phospholipase C from Bacillus cereus. The effect of urea on the enzyme

Abstract

When heated in 8 M-urea, phospholipase C (EC 3.1.4.3) from B. cereus undergoes conformational transitions depending on the temperatures used. These transitions were studied by examining protein fluorescence, iodide quenching of protein fluorescence, UV difference spectroscopy, chemical availability of histidine residues in the enzyme, circular dichroism and catalytic activity. Unless simultaneously exposed to elevated temperatures the enzyme appears to be unaffected by 8 M-urea. Removal of the 2 Zn atoms from the enzyme renders phospholipase C very sensitive to denaturation by 8 M-urea as indicated by fluorescence emission spectra and circular dichroism. Both the native and the Zn-free enzymes are markedly more resistant to irreversible thermal inactivation in the presence of 8 M-urea than in its absence. The response of the enzyme to 8 M-urea and the role of Zn in stabilizing the enzyme are discussed.