RESUMO
This study reports the electrochemical profiling of Nippostrongylus brasiliensis acetylcholinesterase (AChE) wild-type and mutant proteins. An irreversible oxidation signal of electro-active tyrosine (Y), tryptophan (W) and cysteine (C) residues in five mutant proteins along with the wild-type AChE were detected using square-wave voltammetry (SWV) on screen-printed carbon electrodes. Significant differences were observed in the W303L, T65Y and M301W substituted proteins showing a 25-35% higher peak current intensity compared to the Y349Y and F345Y mutants. It was predicted that AChE substituted with electrochemically active residues would produce the greatest signals and this trend was observed in the T65Y, M301W and Y349L mutants. However, conformational changes in the proteins structure as a result of the substitutions appeared to be most influential on peak current intensities. This was demonstrated by the W303L and F345Y mutant enzymes. The current intensity of W303L was greatest despite the removal of its electro-active W residue whereas the F345Y mutant had the lowest peak value despite the addition of an electro-active Y residue. The preliminary results of this study demonstrate that SWV provides a promising tool to probe the presence of electro-active amino acid residues on the surface of a protein produced through bioengineering.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/genética , Substituição de Aminoácidos , Eletroquímica/métodos , Engenharia de Proteínas , Animais , Carbono/química , Eletroquímica/instrumentação , Eletrodos , Ligantes , Modelos Moleculares , Nippostrongylus/enzimologia , Conformação ProteicaRESUMO
The electrochemical oxidation of the benzothiazole dye Thioflavin T (ThT) was found to be modulated by its interaction with electric eel acetylcholinesterase (AChE). Modifications of AChE by trace amounts of small molecule inhibitors such as carbachol and paraoxon were detectable electrochemically using minimal reagents and with greater sensitivity than attainable through conventional fluorescence approaches. This property appears to be unique to ThT, since its closely related neutral derivative BTA-1 only interacts with AChE, but is not significantly affected by the presence of small molecule inhibitors.
Assuntos
Acetilcolinesterase/metabolismo , Técnicas Eletroquímicas/métodos , Tiazóis/metabolismo , Acetilcolinesterase/química , Benzotiazóis , Sítios de Ligação , Carbacol/química , Carbacol/farmacologia , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Eletrodos , Oxirredução , Paraoxon/química , Paraoxon/farmacologia , Espectrometria de Fluorescência/métodos , Tiazóis/químicaRESUMO
The detection of trace levels of paraoxon and carbofuran was achieved utilizing differential pulse voltammetry (DPV) on gold disposable electrochemical printed (DEP) chips. The nanostructured gold surface of the chips enables highly sensitive oxidation of the thiocholine (TCh) product even in the absence of costly surface modifications. The inhibition of AChE activity at varying insecticide concentrations was detected with low detection limits of 10 ppb (36 nM) for paraoxon and 8 ppb (18 nM) for carbofuran. Fine-tuning of the experimental conditions will allow for the application of unmodified DEP gold chips for inexpensive on-field detection of AChE inhibition by various insecticides at or below the allowable concentrations set by European and North American regulation standards.
