title: Voltammetric detection of ovalbumin using a peptide labeled with an electroactive compound.
abstract: For this study, a new method was developed to electrochemically detect ovalbumin via its binding with the peptide-1(RNRCKGTDVQAW) in lysozymes. The peptide that exists at the C-terminal of a lysozyme was combined with ovalbumin. When an electroactive compound was introduced to the N-terminal side of the peptide through ethylene gycolbis(sulfosuccinimidyl succinate), the labeled peptide-1 served as a probe for the detection of ovalbumin. The electrode responses of labeled peptide-1 were measured after the labeled peptide-1 and ovalbumin were incubated in a 0.1 M phosphate buffer (pH 5.6). As a result, the electrode response decreased as the concentration of ovalbumin increased. The detection limit of ovalbumin was 2.3 × 10(-11) M as estimated at 3-fold the standard deviation (3σ) (n = 5). Because the steric structure of the peptide and some of the amino acid residues were related to the binding, we prepared a peptide-2, to which the N- and C-terminals of peptide-1 were alternated. The decrease in the response for the labeled peptide-2 was less than that for the labeled peptide-1. In addition, the peak current of a peptide-3, for which the D of peptide-1 was replaced with S, was hardly changed with or without ovalbumin. Therefore, it was clear that the binding was influenced by the steric factors and by the sequence of the peptide. However, a peptide-1 with bis(sulfosuccinimidyl) suberate was designed to investigate the hydrophobic influences on the probe. The change in the peak current was smaller than that of peptide-1 with ethylene gycolbis(sulfosuccinimidyl succinate), which was due to the hydrophobic properties of the alkyl chain between the peptide and the ovalbumin. The proposed method could be applied to the determination of ovalbumin in egg whites. Consequently, the concept becomes an electrochemical sensing method for proteins based on the protein-peptide interaction.
authors.affiliation: Maebashi Institute of Technology, Gunma 371-0816, Japan. Electronic address: kzsuga@maebashi-it.ac.jp.
authors.forename: Kazuharu
authors.initials: K
authors.lastname: Sugawara
authors.affiliation: Maebashi Institute of Technology, Gunma 371-0816, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kadoya
authors.affiliation: Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Toyama 930-8555, Japan. Electronic address: hkraatz@uwo.ca.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kuramitz
authors.affiliation: Laboratory of Environmental Remediation, Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Shunitz
authors.initials: S
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003630:Daunorubicin / Q000737:chemistry*; D004529:Egg White / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D057230:Limit of Detection; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D010047:Ovalbumin / Q000032:analysis*; D010455:Peptides / Q000737:chemistry*; D011784:Quail
chemical_list: D010455:Peptides; D010047:Ovalbumin; D009113:Muramidase; D003630:Daunorubicin
keywords: Daunomycin; Labeling; Lysozyme; Ovalbumin; Peptide