The role of the aminophospholipid, phosphatidylethanolamine (PE), has been well established to act as a non-protein molecular chaperone in the folding and assembly of polytopic membrane proteins. However, such studies with soluble proteins have not been done so far and in particular with the heme proteins. We have used the heme enzyme, horseradish peroxidase (HRP), as the model heme protein and studied the effect of different phospholipids on its refolding from denatured state. Dimyristoylphosphatidyleth-anolamine (DMPE), a bilayer-forming PE, was able to increase the reactivation yield of denatured HRP upon 30 min refolding at 25degreesC. However, dioleoylphosphatidylethanolamine (DOPE), containing one double bond in the fatty acid chains, which does not favour bilayer organization, did not support proper refolding. The phospholipids with N-methylated head groups, phosphatidylcholines, e.g., DMPC and DOPC showed differential effects when DMPC remained mostly non-supportive while DOPC on the contrary led to inhibition of the refolding of the denatured heme enzyme. Fluorescence spectroscopic studies also indicated changes in the microenvironments of the heme moiety and the single tryptophan residue of HRP in presence of the aminophospholipid. (C) 2003 Elsevier Science (USA). All rights reserved.