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Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

[Image: see text] Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enable...

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Autores principales: Gattu, Srikanth, Crihfield, Cassandra. L., Holland, Lisa A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2016
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214287/
https://www.ncbi.nlm.nih.gov/pubmed/27936604
http://dx.doi.org/10.1021/acs.analchem.6b04074
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author Gattu, Srikanth
Crihfield, Cassandra. L.
Holland, Lisa A.
author_facet Gattu, Srikanth
Crihfield, Cassandra. L.
Holland, Lisa A.
author_sort Gattu, Srikanth
collection PubMed
description [Image: see text] Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (K(M)) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K(M) value of 3.3 ± 0.8 mM (V(max), 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K(M) of 2 ± 1 mM (V(max), 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K(M) value of 3 ± 2 mM (V(max), 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of V(max), the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages.
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spelling pubmed-52142872017-01-09 Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage Gattu, Srikanth Crihfield, Cassandra. L. Holland, Lisa A. Anal Chem [Image: see text] Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (K(M)) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K(M) value of 3.3 ± 0.8 mM (V(max), 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K(M) of 2 ± 1 mM (V(max), 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K(M) value of 3 ± 2 mM (V(max), 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of V(max), the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. American Chemical Society 2016-12-12 2017-01-03 /pmc/articles/PMC5214287/ /pubmed/27936604 http://dx.doi.org/10.1021/acs.analchem.6b04074 Text en Copyright © 2016 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Gattu, Srikanth
Crihfield, Cassandra. L.
Holland, Lisa A.
Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title_full Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title_fullStr Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title_full_unstemmed Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title_short Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage
title_sort microscale measurements of michaelis–menten constants of neuraminidase with nanogel capillary electrophoresis for the determination of the sialic acid linkage
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214287/
https://www.ncbi.nlm.nih.gov/pubmed/27936604
http://dx.doi.org/10.1021/acs.analchem.6b04074
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