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

Paper ID Volume ID Publish Year Pages File Format Full-Text
21278 43214 2012 6 PDF Available
Title
Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination
Abstract

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.

Keywords
Limulus amebocyte lysate; Coagulogen; Endotoxin; Microbeads; Aggregation
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Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 113, Issue 3, March 2012, Pages 406–411
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us