title: Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents.
abstract: The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC(50), under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC(50)=3.7 × 10(-6)M, K(i)=8.6 × 10(-7)M), ascorbic acid (IC(50)=1.2 × 10(-5)M), benzoic acid (IC(50)=7.2 × 10(-5)M, K(i)=2.0 × 10(-5)M) and azelaic acid (IC(50)=1.3 × 10(-4)M, K(i)=4.2 × 10(-5)M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.
authors.affiliation: University of Medicine and Pharmacy, Faculty of Pharmacy, 400012 Cluj-Napoca, Romania.
authors.forename: Veronica Harceaga
authors.initials: VH
authors.lastname: Sima
authors.forename: Stephanie
authors.initials: S
authors.lastname: Patris
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Aydogmus
authors.forename: Ahmad
authors.initials: A
authors.lastname: Sarakbi
authors.forename: Robert
authors.initials: R
authors.lastname: Sandulescu
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Kauffmann
mesh_terms: D000363:Agaricales / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods*; D057886:Bleaching Agents / Q000032:analysis* / Q000494:pharmacology; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008280:Magnetics; D008543:Melanins / Q000096:biosynthesis; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D015203:Reproducibility of Results; D012867:Skin / Q000187:drug effects* / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
chemical_list: D057886:Bleaching Agents; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008543:Melanins; D019809:Streptavidin; D014442:Monophenol Monooxygenase