Studies on the interaction of Rhizopus arrhizus lipase with dipalmitoylphosphatidylcholine liposomes

Abstract

The interaction of Rhizopus arrhizus lipase with phospholipid vesicles was investigated in order to better understand the nature of lipase interfacial processes. The relative electrophoretic mobility of Rhizopus arrhizus lipase bound to dipalmitoylphosphatidylcholine (DPPC) vesicles through 1% agarose gels decreased in a sigmoidal (cooperative) fashion as the DPPC concentration increased, with an apparent dissociation constant ( K d,app) of 3.79 mM [DPPC] (Δ G ° b,app= −3.30 kcal mol −1). This phenomenon was not observed with dioleylphosphatidylglycerol (DOPG) vesicles, suggesting the absence of binding of the lipase to these liposomes. Quasi-elastic light scattering studies of these interactions showed that DPPC vesicles with an average diameter of 144 and 172 nm increased in size in a sigmoidal (cooperative) fashion as a function of increasing lipase concentration, with K d,app values for these interactions of 6.92 × 10 −7 M [lipase] (Δ G ° b,app= −8.4 kcal mol −1) and 2.35 × 10 −7 M [lipase] (Δ G ° b,app= −9.05 kcal mol −1), respectively. The lipase, however, did not bind to 116 nm DPPC vesicles or to DOPG vesicles of any size. Data for the lipase-DPPC interactions were fitted to a Hill model for determination of dissociation constants K d,app and cooperativity indices (Hill coefficients). The Hill coefficients derived were 5.0 and 2.4 for the 144 and 172 nm vesicles, and 13.0 for the band retardation assay, respectively. Binding of the lipase to DPPC vesicles abolished their characteristic lipid phase transition at 42°C as determined by fluorescence polarization spectroscopy using 1,6-diphenylhexatriene as a probe. Binding lead to changes in the secondary structure of the lipase which included slight increases in the α-helical content as determined by circular dichroism spectroscopy.