Electrophoretic detection of chitinase isoenzymes using the PhastSystem.
Zymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter-staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4-methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4-methylumbelliferone). The pIs of chitinase isoenzymes were estimated by using coloured pI markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii.
McBride
J D
JD
Wellcome Research Laboratories, Beckenham, Kent.
Stubberfield
C R
CR
Hayes
D J
DJ
D000818:Animals; D002688:Chitinases / Q000032:analysis*; D005456:Fluorescent Dyes; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000032:analysis*; D008168:Lung / Q000201:enzymology*; D011020:Pneumonia, Pneumocystis / Q000201:enzymology*; D051381:Rats
D005456:Fluorescent Dyes; D007527:Isoenzymes; D002688:Chitinases