Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination

The effects of thermal treatment on Limulus amebocyte lysate (LAL) reagent were studied. Thermal resistances of enzymes and coagulogen in LAL reagent were evaluated by aggregometry and SDS-PAGE. Although enzyme activities of LAL reagent were completely lost after heating at temperatures above 60 °C for 10 min, gelating activities of coagulogen were retained even over 80 °C. Phenylmethanesulfonyl fluoride (PMSF; 1 mmol/mL), a strong non-specific serine-protease inhibitor, did not completely inactivate serine-protease activities of LAL. As a result, complete hydrolysis of coagulogen to coagulin was unexpectedly obtained. Solvent treatment of LAL was similar in effect to thermal treatment of LAL, but there were 2 problems: complete removal of solvent from samples and increased solution turbidity during preparation. To study the application of thermal-treated LAL, we conjugated it with titania particles. LAL-conjugated titania particles were obtained as small aggregates between titania nanoparticles and thermal-treated LAL (LAL-conjugated microbeads; LCM). When the mixture of LCMs and fresh LAL reagent was reacted with endotoxin an acute aggregation of LCMs was induced prior to the aggregate formation of LAL as monitored by stirring turbidimetry. This method, endotoxin microbeads aggregometry (EMA) may provide a rapid and sensitive method for endotoxin determination.