Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins.
Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.
Petsch
D
D
GBF, Gesellschaft für Biotechnologische Forschung, Bioverfahrenstechnik, Braunschweig, Germany.
Deckwer
W D
WD
Anspach
F B
FB
D000327:Adsorption; D001427:Bacterial Toxins / Q000032:analysis* / Q000302:isolation & purification; D019286:Endopeptidase K; D004731:Endotoxins / Q000032:analysis* / Q000302:isolation & purification; D004800:Enzymes, Immobilized; D004926:Escherichia coli; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008033:Limulus Test / Q000379:methods*; D008565:Membrane Proteins; D009113:Muramidase; D015203:Reproducibility of Results
D001427:Bacterial Toxins; D004731:Endotoxins; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008565:Membrane Proteins; C025357:endotoxin binding proteins; D009113:Muramidase; D019286:Endopeptidase K