Unusual Thermal Stability of Human Secreted Phospholipase A2 Enzymes

Abstract

Analysis of the thermal stability of proteins in general and enzymes in particular is important for understanding their molecular mechanisms and for their analytical or industrial exploitations. While enzymes with extreme thermostability (resistance to irreversible inactivation at high temperatures) have been identified in hyperthermophilic microorganisms, their occurrence in mammals is rare. Here, we have studied the thermostability of two human secreted phospholipase A2 (PLA2) enzymes, namely, group IB PLA2 and group IIA PLA2. In both cases, the enzymes exhibited maximum phospholipid hydrolyzing activity between 60 and 70oC. [The target membranes contained 70% phosphatidylcholine (PC) and 30% phosphatidylglycerol (PG), and the buffer contained 50 mM Hepes, pH 7.4, and 2 mM CaCl2).] Parallel circular dichroism (CD) measurements identified sigmoidal temperature dependencies of the ellipticity at 222 nm with “melting” transition temperatures around 90-95oC. The activity rapidly declined at higher temperatures and was practically absent between 85 and 100oC. However, when the sample containing the enzyme and the substrate (PC/PG membranes) was “cooked” at 100oC for up to 30 minutes and then cooled to favorable temperatures (e.g., 40-50oC), the activity of the enzyme was gradually recovered, reaching even higher levels than those initially measured at those temperatures. This was accompanied with partial restoration of the CD spectra, hence the secondary structure of PLA2s. These facts indicate an unusual thermostability of human group IB and IIA PLA2s, which may be supported by a high content of intramolecular disulfide bonds in these enzymes (7 disulfides per 124-126 residues). We interpret these data in terms of partial thermal dissociation of the disulfides at near-boiling temperatures and their restoration upon cooling. Experimental verification of this hypothesis, as well as quantitative characterization of the thermodynamic and kinetic parameters of the thermostability of the enzymes, are in progress.