Bacterial immunoglobulin A proteases monitored by continuous spectrophotometry.
IgA proteases were estimated in a turbid aqueous two-phase system with 10% polyethylene glycol-Tris buffer, where IgA spontaneously concentrates in microscopic spherical particles (less than 1 micron). After enzymatic cleavage of IgA into Fab alpha and Fc alpha fragments, these fragments are soluble and decreasing turbidity is observed. The reaction may be followed by conventional spectrophotometry. In this manner, IgA proteases may be estimated in 10 min. Examples of the utility of the method are given with results from inhibitor studies, estimation of Km and purification of IgA protease from Haemophilus influenzae.
D001322:Autoanalysis; D006193:Haemophilus influenzae / Q000201:enzymology; D007128:Immunoglobulin Fragments / Q000032:analysis; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D012995:Solubility; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D007128:Immunoglobulin Fragments; D011480:Protease Inhibitors; D011092:Polyethylene Glycols; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase