We present astudy of lipid monolayer hydrolysis at constant area (isochoric method) toassess the optimal lateral surface pressure value (Π_opt) andthus, the surface packing density of the lipid, at which the activity of agiven lipolytic enzyme is maximal. This isochoric method consists of ameasurement of the decrease down to zero of the Π_opt ofphospholipid substrate monolayer due to continuous hydrolysis using only onereaction compartment. We performed the comparison of both approaches usingseveral commercially available and literature-evaluated sPLA₂s.Also, we characterized for the first time the profile of hydrolysis of DLPCmonolayers catalyzed by a sPLA₂ from Streptomyces violaceoruberand isoenzymes purified from Bothrops diporus venom. One of theseviper venom enzymes is a new isoenzyme, partially sequenced by a massspectrometry approach. We also included the basic myotoxin sPLA₂-IIIfrom Bothrops asper. Results obtained with the isochoric method and thestandard isobaric one produced quite similar values of Π_opt,validating the proposal. In addition, we propose a new classificationparameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN.m^-1and 10 mN.m^-1, termed here as LR_20/10 index. This indexdifferentiates quite well ?high surface pressure? from ?low surface pressure?sPLA₂s and, by extension; it can be used as afunctional criterion for the qualityof a certain enzyme. Also, this index could beadded to the grouping systematic criteria for the superfamily proposed forphospholipase A₂.

Acknowledgementsand Support This work was supported by grants from SECyT-UNC,FONCYT-MinCyT and CONICET, Argentina, as well as from International Centre forGenetic Engineering and Biotechnology (ICGEB CRP/COS13-01) and Vicerrectoría deInvestigación, Universidad de Costa Rica (UCR 741-B4-100). BL and GDFespecially wish to thank the Ibero-American Network CYTED BIOTOX (Red Biotox212RT0467) for the support given to research groups involved in the network.