Anal. Chem. 2003, 75, 3681-3687
Articles
A Continuous-Flow System for High-Precision
Kinetics Using Small Volumes
Xianzhi Zhou,† Rohit Medhekar,‡ and Michael D. Toney*
Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, California 95616
There is a long history of analyzing chemical and enzyme
A generally applicable continuous-flow kinetic analysis
system that gives data of a precision high enough to
measure small kinetic isotope effects for enzymatic and
nonenzymatic reactions is described. It employs com-
mercially available components that are readily assembled
into an apparatus that is easy to use. It operates under
laminar flow conditions, which requires that the time
between the initiation of the reaction in the mixer and the
observation be long enough that molecular diffusion can
effect a symmetrization of the concentration profile that
results from a thin plug of reagents introduced at the
mixer. The analysis of a second-order irreversible reaction
under pseudo-first-order conditions is presented. The
Yer sin ia pestis protein tyrosine phosphatase catalyzed
hydrolysis of p-nitrophenyl phosphate is characterized
with the system, and a proton inventory on kcat is pre-
sented.
kinetics using flow techniques.2-4 These fall into three classes:
stopped flow, quenched flow, and continuous flow. It has generally
been assumed that the applicability of flow techniques to kinetic
analyses requires that the flow of the liquid through the tubing
of the instrument be turbulent.2,3 This ensures there is no radial
variation in the flow rate about the cental axis of the flow (i.e.,
there is uniform flow). When this is true, one can calculate the
reaction time of a given volume element simply from the total
flow rate and the distance traveled from the mixer.
Recently, several papers have appeared that report the suc-
cessful analysis of protein folding and enzyme kinetics using
conditions in which turbulent flow is not achieved.5-10 Instead,
laminar flow prevails, where there is a radial variation of flow rate
about the central axis of the flow. Laminar flow, unlike turbulent
flow, has the effect of parabolically distorting the flow profile. The
success of the reported kinetic analyses under laminar flow
conditions and a recent computational analysis of the effect of
laminar flow and molecular diffusion on such experiments
demonstrates that its presence is not prohibitive to accurate kinetic
analyses.11
A continuous-flow system that operates under laminar flow
conditions is described here. It is a generally applicable system
that can be used to analyze both enzymatic and nonenzymatic
reactions. The components are commercially available, and it is
straightforward to use to generate high precision kinetic data
useful in kinetic isotope effect as well as other determinations.
Steady-state spectrophotometric enzyme kinetic assays are
typically performed by mixing solutions that differ in substrate
concentration in several cuvettes and observing the initial rates
of the individual reactions in a spectrophotometer. Nonlinear
regression analysis of the initial rate data typically yields kinetic
parameters that have errors of 5-20%. This is generally acceptable
for many routine kinetic analyses, but some require much higher
precision. Kinetic isotope effect (KIE) measurements are a good
example. Primary deuterium KIEs are generally large enough
(e.g., 2-6-fold) that kinetic measurements using manual tech-
niques define them sufficiently well. On the other hand, secondary
deuterium and heavy atom KIEs pose greater challenges that
cannot generally be met by manual techniques. Rosenberg and
Kirsch have shown that with extreme care, these challenges are
not insurmountable.1 The development of a general purpose, easy
to use instrument capable of measuring kinetics with high
precision would be a welcome addition to the armamentarium of
the biochemist and chemist alike.
EXPERIMENTAL SECTION
Instrumental Setup. The continuous-flow system is dia-
gramed in Figure 1. It employs three syringe pumps that were
purchased from KD Scientific (New Hope, PA). The two pumps
(2) Johnson, K. A. Methods Enzymol. 1 9 9 5 , 249, 38-61.
(3) Hartridge, H.; Roughton, F. J. W. Proc. R. Soc. (London) 1 9 2 3 , A104, 376-
394.
(4) Gibson, Q. H. Methods Enzymol. 1 9 6 9 , 16, 187-228.
(5) Zhou, X. Z.; Toney, M. D. J. Am. Chem. Soc. 1 9 9 8 , 120, 13282-13283.
(6) Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas, D. J. Biochemistry
1 9 9 8 , 37, 7664-7669.
* To whom correspondence should be addressed. Phone: 530-754-5282.
Fax: 530-752-8995. E-mail: mdtoney@ucdavis.edu.
† Current address: Monsanto Company, 800 North Lindbergh Boulevard, St.
Louis, MO 63167.
‡ Current address: National Enzyme Company, 15366 US Hwy 160, Forsyth,
MO 65653.
(7) Simmons, D. A.; Konermann, L. Biochemistry 2 0 0 2 , 41, 1906-1914.
(8) Teilum, K.; Maki, K.; Kragelund, B. B.; Poulsen, F. M.; Roder, H. Proc. Natl.
Acad. Sci. U.S.A. 2 0 0 2 , 99, 9807-9812.
(9) Park, S. H.; Shastry, M. C.; Roder, H. Nat. Struct. Biol. 1 9 9 9 , 6, 943-947.
(10) Kuwata, K.; Shastry, R.; Cheng, H.; Hoshino, M.; Batt, C. A.; Goto, Y.; Roder,
H. Nat. Struct. Biol. 2 0 0 1 , 8, 151-155.
(1) Rosenberg, S.; Kirsch, J. F. Anal. Chem. 1 9 7 9 , 51, 1379-1383.
(11) Konermann, L. J. Phys. Chem. 1 9 9 9 , 103, 7210-7216.
10.1021/ac034068j CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/29/2003
Analytical Chemistry, Vol. 75, No. 15, August 1, 2003 3681