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429 are conjugated to each QD. However, this number is probably far less for
430 bulky enzymes like AChE compared to HEWL owing to surface crowding
431 and steric effects.
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490
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The dark luminescence bands in the agarose gel reflect the high ex-
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433 tinction coefficient and luminescence quantum yield of QDs on expo-
434 sure to ultraviolet wavelengths. The poor separation between bands in
435 Fig. 6A and B is not a surprise with QDs on gel electrophoresis as previ-
436 ous reports indicate [71,73,74]. QDs are large molecules with molecular
437 weights near ~400 kDa. In addition, each streptavidin molecule is
438 around 75 kDa [75], which gives a total molecular weight to be around
439 ~925 kDa (considering an average of 7 streptavidin molecules per QD).
440 This large molecular weight of QD in comparison to enzymes (14–
441 280 kDa) probably accounts for the poor separation between bands ob-
442 served in the gels. The negligible movement of AChE–QD conjugates is
443 accounted by large size of tetrameric enzyme (~280 kDa), making
444 QD–Enz conjugates too large for entry into the pores of the agarose gel.
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Although we have not measured the exact number of enzymes con-
446 jugated to QD surface, increased level of enzyme biotinylation raises the
447
448
449
450
451
452
453
packing density of enzymes in QD surface as confirmed by QD lumines-
cence data. Hence, the observation of complete enzyme activity in
QDEnzBio with bulky macromolecules like AChE and ALP (Figs. 3 and
4) at all biotinylation levels indicates that the packing density of enzymes
on QD surface is moderate enough to permit enzyme to retain native-like
activity. Higher packing densities may be needed to start observing de-
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In this study, the effect of biotinylation and conjugation of hydrolytic
enzymes HEWL, ALP and AChE to QDs was investigated. All three en-
zymes revealed a negligible change in their catalytic activity upon conju-
gation to QDs via streptavidin–biotin linkage. In the case of biotinylated
HEWL, the enzyme activity was profoundly affected by degree of biotinyl-
ation and salt concentration in the medium but this was irrespective of
conjugation to the QD. The stability (luminescence) of QDs was preserved
in all QD–Enz conjugates. Thus, we conclude that the streptavidin–biotin
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We thank Prof. Vinod Subramaniam from FOM Institute AMOLF for
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The Michaelis–Menten kinetics of HEWL, ALP and AChE; absorbance
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472 vs. time traces of all enzyme catalyzed hydrolysis reactions; emission
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Supplementary data to this article can be found online at http://dx.
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Please cite this article as: A. Iyer, et al., Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity, Biochim. Biophys.