Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern.
Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.
Zhang
Zhenqing
Z
Departments of Chemistry and Chemical Biology and Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.
Park
Youmie
Y
Kemp
Melissa M
MM
Zhao
Wenjing
W
Im
A-Rang
AR
Shaya
David
D
Cygler
Miroslaw
M
Kim
Yeong Shik
YS
Linhardt
Robert J
RJ
D001173:Arthrobacter / Q000201:enzymology; D001439:Bacteroides / Q000201:enzymology; D019765:Chondroitin ABC Lyase / Q000378:metabolism*; D002808:Chondroitin Lyases / Q000378:metabolism*; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D013058:Mass Spectrometry; D011514:Proteus vulgaris / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014783:Viscosity
D011994:Recombinant Proteins; D002808:Chondroitin Lyases; D019765:Chondroitin ABC Lyase