Directional degradation of beta-chitin by chitinase A1 revealed by a novel reducing end labelling technique.
A novel procedure for labelling the molecular ends of beta-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of beta-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the beta-chitin microcrystals, and also to label the reducing tips of beta-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.
Imai
Tomoya
T
Wood Research Institute, Kyoto University, Uji, Japan.
Watanabe
Takeshi
T
Yui
Toshifumi
T
Sugiyama
Junji
J
D000818:Animals; D001407:Bacillus; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D020134:Catalytic Domain / Q000502:physiology; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002688:Chitinases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D003460:Crystallization; D003461:Crystallography / Q000379:methods; D017979:Gold Colloid / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity / Q000502:physiology
D017979:Gold Colloid; D015335:Molecular Probes; D011134:Polysaccharides; D002686:Chitin; C053311:biotin hydrazide; D001710:Biotin; D019809:Streptavidin; D002688:Chitinases