Biochimica et Biophysica Acta 2014-09-01

Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity.

Aditya Iyer, Anil Chandra, Rajaram Swaminathan

Index: Biochim. Biophys. Acta 1840(9) , 2935-43, (2014)

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Abstract

Tagging a luminescent quantum dot (QD) with a biological like enzyme (Enz) creates value-added entities like quantum dot-enzyme bioconjugates (QDEnzBio) that find utility as sensors to detect glucose or beacons to track enzymes in vivo. For such applications, it is imperative that the enzyme remains catalytically active while the quantum dot is luminescent in the bioconjugate. A critical feature that dictates this is the quantum dot-enzyme linkage chemistry. Previously such linkages have put constraints on polypeptide chain dynamics or hindered substrate diffusion to active site, seriously undermining enzyme catalytic activity. In this work we address this issue using avidin-biotin linkage chemistry together with a flexible spacer to conjugate enzyme to quantum dot.The catalytic activity of three biotinylated hydrolytic enzymes, namely, hen egg white lysozyme (HEWL), alkaline phosphatase (ALP) and acetylcholinesterase (AChE) was investigated post-conjugation to streptavidin linked quantum dot for multiple substrate concentrations and varying degrees of biotinylation.We demonstrate that all enzymes retain full catalytic activity in the quantum dot-enzyme bioconjugates in comparison to biotinylated enzyme alone. However, unlike alkaline phosphatase and acetylcholinesterase, the catalytic activity of hen egg white lysozyme was observed to be increasingly susceptible to ionic strength of medium with rising level of biotinylation. This susceptibility was attributed to arise from depletion of positive charge from lysine amino groups after biotinylation.We reasoned that avidin-biotin linkage in the presence of a flexible seven atom spacer between biotin and enzyme poses no constraints to enzyme structure/dynamics enabling retention of full enzyme activity.Overall our results demonstrate for the first time that streptavidin-biotin chemistry can yield quantum dot enzyme bioconjugates that retain full catalytic activity as native enzyme.Copyright © 2014 Elsevier B.V. All rights reserved.