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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #61163
Title: SILVER STAIN DETECTION OF CHITINOLYTIC ENZYMES AFTER POLYACRYLAMIDE GEL

Author
item MAREK, STEPHEN - UNIV OF CA-DAVIS
item ROBERTS, CRAIG - UNIV OF MO
item Beuselinck, Paul
item KARR, ARTHUR - UNIV OF MO

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Chitin is the main structural component of most cell walls of fungi, and the bodies of insects, crabs, and shrimp. Specialized proteins known as chitinolytic enzymes can digest chitin into the smaller building blocks that chitin is made of. Enzymes that can digest chitin are known as chitinases. Bacteria, fungi, plants and animals produce chitinase. Some plants that produce chitinase also are able to resist infections caused by fungi. This paper reports the development of a new laboratory technique that can be used to test for chitinase. Other laboratory techniques to identify chitinase are available, but they have drawbacks. The new silver- staining technique described in this paper solves some of the problems of previous techniques, and it is less expensive. The silver-stained gels can be dried to provide a permanent record of the results and can be digitally recorded and stored electronically for easy retrieval. Silver staining of gels allows more precise and sensitive detection of chitinases than previous techniques.

Technical Abstract: Chitinolytic enzymes catalyze the hydrolysis of chitin, a beta-1,4-linked polymer of N-acetyl-D-glucosamine, which is the main structural component of most fungal cell walls and arthropod exoskeletons and integuments. Chitinases (EC 3.2.1.1 4) and lysozymes (EC 3.2.1.1 7) catalyze the hydrolysis of chitin (and/or peptidoglycan) into oligomers, while beta-N- acetylhexoseaminidases (EC 3.2.1.52) release monomers. Chitinases are typically located after electrophoresis by taking advantage of their ability to locally solubilize glycol chitin from the gel. The remaining glycol chitin is then stained with 0.01% (w/v) Calcofluor white M2R. While this procedure can be used to identify proteins with chitinase activity, it has several drawbacks. We report adaptation of a silver stain method to stably stain glycol chitin in situ allowing visualization of the lytic zones with higher resolution and sensitivity. The stained gels can be dried providing a permanent record of the results. Gels can be analyzed using a normal gel scanner and can be digitally recorded and stored electronically for easy retrieval. The procedure has the advantage of using only chemicals which would normally be present in a precise and sensitive detection of chitinolytic enzymes than Calcofluor white M2R and facilitates the preservation of stained gels by drying.