A rapid and sensitive method for screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay.
OBJECTIVE
The development of a simple, rapid and sensitive method for the screening of chitinase inhibitors using Ostazin Brilliant Red labelled chitin as a substrate for chitinase assay.


RESULTS
The colloidal substrate prepared from the Ostazin Brilliant Red labelled chitin was used in the estimation of chitinase activity with and without the addition of potential chitinase inhibitor. The dye labelled N-acetylglucosamine (GlcNAc) released because of hydrolysis of the substrate was measured spectrophotometrically at 530 nm. The release of dye labelled GlcNAc from the colloidal Red chitin was proportional to the chitinase activity measured using other methods. The activities measured using acid swollen chitin and -Red chitin were comparable in the range 0.1-0.5 U ml(-1). The addition of chitinase inhibitor(s) (1000 ppm) proportionally reduced the release of dye labelled GlcNAc from the substrate in the reaction mixture.


CONCLUSIONS
The assay for chitinase activity measuring the release of dye-labelled GlcNAc was simple, rapid and sensitive. There was no need to measure GlcNAc using a separate colorimetric method.


CONCLUSIONS
The method provides a simple, one-step procedure for quantitative estimation of total chitinolytic activity and for screening of endochitinase and/or N-acetylglucosaminidase inhibitors acting singly or in combination. The method has a great potential in developing medium throughput procedure for the screening of chitinase inhibitors using 96-well plate reader.
Shirazi
F
F
Biochemical Sciences Division, National Chemical Laboratory, Pune 411008, India.
Kulkarni
M
M
Deshpande
M V
MV
D000117:Acetylglucosamine / Q000032:analysis; D002686:Chitin / Q000737:chemistry*; D002688:Chitinases / Q000037:antagonists & inhibitors*; D002855:Chromatography, Thin Layer; D003102:Colloids / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D006868:Hydrolysis; D013379:Substrate Specificity; D014227:Triazines / Q000737:chemistry*
D003102:Colloids; D004791:Enzyme Inhibitors; D014227:Triazines; D002686:Chitin; C011535:procion brilliant red H 3B; D002688:Chitinases; D000117:Acetylglucosamine