Improved fluorescent labeling of chitin oligomers: Chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions.
Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing. AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH 8. Here we analyzed chitin degradation by AMCase in weak acidic to neutral conditions by fluorophore-assisted carbohydrate electrophoresis established originally for oligosaccharides analysis. We found that specific fragments with slower-than-expected mobility as defined by chitin oligosaccharide markers were generated at pH 5.0∼8.0 as by-products of the reaction. We established an improved method for chitin oligosaccharides suppressing this side reaction by pre-acidification of the fluorophore-labeling reaction mixture. Our improved method specifically detects chitin oligosaccharides and warrants quantification of up to 50nmol of the material. Using this strategy, we found that AMCase produced dimer of N-acetyl-d-glucosamine (GlcNAc) at strong acidic to neutral condition. Moreover, we found that AMCase generates (GlcNAc)2as well as (GlcNAc)3under physiological conditions.
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Satoshi
S
Wakita
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Masahiro
M
Kimura
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Naoki
N
Kato
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Akinori
A
Kashimura
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Shunsuke
S
Kobayashi
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Naoto
N
Kanayama
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Misa
M
Ohno
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Shotaro
S
Honda
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Masayoshi
M
Sakaguchi
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan.
Yasusato
Y
Sugahara
Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA; Bioinova Ltd., Prague 142 20, Czechia.
Peter O
PO
Bauer
Department of Chemistry and Life Science, Kogakuin University, Hachioji, Tokyo, Japan. Electronic address: f-oyama@cc.kogakuin.ac.jp.
Fumitaka
F
Oyama
D000818:Animals; D002686:Chitin / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry*; D005453:Fluorescence; D005944:Glucosamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D051379:Mice
D002686:Chitin; D002688:Chitinases; D005944:Glucosamine
Acidic mammalian chitinase; Chitin; Chitin degradation products; Chitin oligomers; Fluorophore; Pre-acidification method