title: Effect of acrylodan conjugation and forced oxidation on the structural integrity, conformational stability, and binding activity of a glucose binding protein SM4 used in a prototype continuous glucose monitor.
abstract: Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H2O2. Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H2O2. An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H2O2. Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: John M
authors.initials: JM
authors.lastname: Hickey
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: Neha
authors.initials: N
authors.lastname: Sahni
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: Rajoshi
authors.initials: R
authors.lastname: Chaudhuri
authors.affiliation: BD Medical - Diabetes Care, Andover, Massachusetts, 01810.
authors.forename: Ajit
authors.initials: A
authors.lastname: D'Souza
authors.affiliation: BD Medical - Diabetes Care, Andover, Massachusetts, 01810.
authors.forename: Andrew
authors.initials: A
authors.lastname: Metters
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: C
authors.initials: C
authors.lastname: Russell Middaugh
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, 66047.
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D015190:Blood Glucose Self-Monitoring / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D000072417:Protein Domains; D055550:Protein Stability
chemical_list: D029968:Escherichia coli Proteins; C038518:acrylodan; D015081:2-Naphthylamine
keywords: acrylodan; chemical modification; glucose; mass spectrometry; oxidation; stability