Enzyme fluorescence spectroscopy

Ha T, Ting A Y, Liang J, Caldwell W B, Deniz A A, Chemla D S, Schultz P G and Weiss S 1999 Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism Proc. Natl Acad. Sc/. USA 96 893-8... 

The protein-containing colloidal solutions of water-in-organic solvents are optically transparent. Hence, absorption spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy are found to be convenient for studying biocatalysis [53]. The reversed micelles are interesting models for studying bioconversion, since the majority of the enzymes in vivo act inside or on the surface of biological membranes. 

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

As the enzyme itself is usually the focus of interest, information on the behavior of that enzyme can be obtained by incubating the enzyme with a suitable substrate under appropriate conditions. A suitable substrate in this context is one which can be quantified by an available detection system (often absorbance or fluorescence spectroscopy, radiometry or electrochemistry), or one which yields a product that is similarly detectable. In addition, if separation of substrate from product is necessary before quantification (for example, in radioisotopic assays), this should be readily achievable. It is preferable, although not always possible, to measure the appearance of product, rather than the disappearance of substrate, because a zero baseline is theoretically possible in the former case, improving sensitivity and resolution. Even if a product (or substrate) is not directly amenable to an available detection method, it maybe possible to derivatize the product with a chemical species to form a detectable adduct, or to subject a product to a second enzymatic step (known as a coupled assay, discussed further later) to yield a detectable product. But, regardless of whether substrate, product, or an adduct of either is measured, the parameter we are interested in determining is the initial rate of change of concentration, which is determined from the initial slope of a concentration versus time plot. 

While it is tempting to explain regulatory and cosolvent effects on the basis of conformational changes favorable or unfavorable to enzyme activity, it is much more difficult to demonstrate the actual involvement, amount, and structural details of such changes. Experimental evidence consists in most cases of bits and pieces provided by techniques such as absorption and fluorescence spectroscopy, circular dichroism, and magnetic circular dichroism. These tools work in solution (and, when desired, at subzero temperatures) to investigate not simply empty enzymes but enzyme—substrate intermediates. However, even with this information, the conformational basis of enzyme activity remains more postulated than demonstrated at the ball and stick level, and in spite of data about the number and sequence of intermediates, definition of their approximate nature, rate constants, and identification of the types of catalysis involved, full explanation of any particular reaction cannot be given and rests on speculative hypothesis. 

Enzyme active sites and receptors rarely interact with hgands without some attendant change in conformation, and the ability to detect and quantify a conformational change hes at the heart of contemporary biochemical kinetics. See Induced Fit Model Fluorescence Spectroscopy Linked Functions Flemoglobin Cooperativity... 

Keywords. Enzymes, Mass spectrometry, IR-thermography, Optical detection. Fluorescence spectroscopy... 

WuXJ, Choi MMF. Optical enzyme-based glucose biosensors. In Geddes CD, Lakowicz JR (Eds), Topics in Fluorescence Spectroscopy, Vol. 11. Springer, New York, 2006, pp. 201-236. 

Ha, T., Ting, A. Y., Liang, J., Caldwell, W. B., Deniz, A. A., Chemla, D. S., Schultz, P. G., and Weiss, S (1999a). Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl. Acad. Sci. USA 96, 893-898. 

The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea/EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and enzyme recovery by membrane filtration give d-pantolactone with 50% yield and 90-95% ee over 6 days. 

The nature of the relation between lipid structure changes and enzyme activation has been investigated using, for example, fluorescence spectroscopy and calorimetry (see Section 3.1). Figure 1.5 shows the effect of temperature on the hydrolytic activi-... 

The enzyme was unfolded by guanidine HC1 treatment through two detectable phases Phases 1 and 2 were observed by fluorescence spectroscopy derived from tryptophan residue.14 The fluorescence maximum moved gradually from 336 to 331 nm as the... 

Westphal AH, Matorin A, Hink MA, Borst JW, van Berkel WJH, Visser AJWG. Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase. J. Biol. Chem. 2006 281 11074-11081. 

Using fluorescence spectroscopy to analyze enzyme- substrate interactions Section 8.3.2 Using irreversible inhibitors to map the active site Section 8.5.2 Using transition state analogs to study enzyme active sites Section 8.5.3 Catalytic antibodies as enzymes Section 8.5.4... 

Fluorescence. The use of molecular fluorescence spectroscopy for the quantitation of enzyme reaction products has resulted in detection limits that are several orders of magnitude lower than those achieved by standard absorbance methods. At low analyte concentrations, fluorescence emission intensity is directly proportional to concentration, and its value depends on both the molar absorptivity of the analyte at the excitation wavelength, and the fluorescence quantum yield of the analyte, under the assay conditions. 

Using fluorescence spectroscopy to analyze enzyme substrate in teracllons (p- 213)... 

The fluorescent spectroscopy techniques were used to observe the interaction of dyes with macromolecules.5 The fluorescent spectra and steady-state fluorescence anisotropy of dyes in the presence of enzymes were determined with an Aminco-Bowman Series 2 spectrofluorimeter (ThermoSpectronic, USA) equipped with polarizers. 

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