Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Abstract

A method for sensitively monitoring enzyme kinetics and activities by using dual-color f luorescence crosscorrelation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two f luorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and f luorophores at each 5 end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K M of 14 ؎ 1 nM and a k cat of 4.6 ؎ 0.2 min ؊1 . In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color f luorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.Kinetic studies on enzymes are among the most important tools for understanding biological interactions at the molecular level. In combination with new approaches in genetic engineering and structure determination, there have been major efforts in recent years to develop more sensitive and precise techniques for characterizing the kinetics of enzyme reactions. These techniques have made it possible to develop efficient assays for analyzing catalytic parameters such as turnover rates, substrate specificity, as well as regio-and stereospecificity; they constitute a major part of the biochemical and pharmaceutical research. Moreover, the alteration of catalytic properties, either by evolutionary biotechnology (1-4) or by rational approaches, is of special interest for medical and industrial applications. The desired features are as follows: accelerated reaction rates; higher, relaxed, or even new substrate specificities; tolerance to nonnatural environments; and novel types of reactions. Modern rational design is the interplay between computer modeling and experimental testing of designed molecules by sensitive and quantitative assays. On the other hand, evolutionary approaches make use of rapid and highly sensitive screening assays to detect minute quantities of better adapted individuals among a large excess of alternatives. For all these purposes, fluorescence correlation spectroscopy (FCS) (5-8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled ...

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

“…Nevertheless, the amplitude of the background crosscorrelation curve is less than 10% of the value of the positive sample. This demonstrates a performance of the setup that is comparable and potentially even superior to one-photon optics (12,14).…”

“…An exciting perspective is given further for intracellular applications of dual-color crosscorrelation analysis, for which two-photon excitation has already been demonstrated to be superior (23), in particular because of its inherent optical sectioning capabilities preventing photodamage in off-focus areas. As previously pointed out (14), the concept of dual-color crosscorrelation can be applied to any association or dissociation process or any reaction where a chemical or physical linkage between two labeled molecules is formed or destroyed, independent of the size of the observed molecule and not limited by a certain distance between the fluorophores, in contrast to other detection methods such as energy transfer approaches.…”

“…Dual-color crosscorrelation analysis (12,13), a conceptual modification of FCS using two spectrally separable fluorescent labels, yields a considerable improvement in signal specificity for heterogeneous systems where molecular interactions of different species are to be observed. The simplicity of the measurement process has recently even encouraged biotechnological applications leading to online kinetics of enzymatic reactions (14) and fast enzyme screening applications (15,16). In principle, any molecular association and dissociation, as well as the formation or cleavage of chemical bonds, can be studied simply by simultaneous detection of two spectrally separated fluorophores, following the amplitude of the crosscorrelation function in real time during the reaction.…”

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

“…FCS, as initially detailed by Magde and coworkers (16)(17)(18), involved measuring the statistical fluctuation in the fluorescence signal at a single point as molecules passed through a fixed laser beam; dynamical information was obtained from these fluctuations. FCS has recently been used to measure diffusion constants of small proteins both in the cell membrane and cytoplasm (19)(20)(21)(22); it works well for measuring rapid processes occurring on the microsecond to millisecond time scale. In contrast, ICS is useful for measuring processes occurring on a slower time scale (seconds to minutes) for spatially distinct objects, such as polyplexes within the cell.…”

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

“…There are many applications of FCCS to detect bimolecular interactions in solution [21][22][23]. However, quantitative measurements of interactions are also important in the living cell environment.…”