The spectrophotometric assay is a classic enzyme test and still remains as the most widely used assay for the low cost and robust reproducibility. During a spectrophotometric assay, the operator follows the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution. Most tests use the UV/visible (UV/vis) spectroscopy as the detection method, which usually falls into the wavelength range of 100-1100 nm. If the light is in the visible region, meaning the wavelength of 400-700 nm or more broadly 360-900 nm, the color of the assay can be visibly captured by naked eyes. Therefore, this type of tests is also called colorimetric assays.
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When the UV/vis spectroscopy is performed to determine the reaction rate or catalysis efficiency, the reaction solution presents color or brightness drifts from reactants to products, and such drifts can be detected by the UV/vis spectrometer. Subsequently, the rate constant of this enzyme reaction can be quantified by measuring the UV/vis absorbance spectrum over a period of time. Often as the first step, the optimal wavelengths for detecting all species involved in the reaction will be determined. The ideal wavelength should clearly indicate the difference between the reactant and product, while interferences from other chemicals stay minimal. Sometimes, more than one wavelength need to be used to produce strong signals to calculate the enzyme activity. For example, the common cofactors NADH and NADPH are frequently used in enzyme activity assays, since they absorb UV light in the reduced forms, and not in the oxidized forms. An oxidoreductase that uses NADH or NADPH as the cofactor can, therefore, be characterized by tracking the decreases in UV absorbance at a wavelength of 340 nm, which is the sign of the cofactor being consumed as the enzyme reaction proceeds.
When measuring the enzyme reaction, the sensitivity of the detector and stability of the whole spectroscopy system are critical to reliable measurement. The first attribute guarantees detection of subtle changes in light intensity of an enzyme reaction. The latter one maintains low fluctuation and noise levels. Jointly, the two aspects of the instrument control the signal-to-noise ratio, and high-quality instruments can test enzyme activities in far more cases than an average instrument.
The colorimetric assay uses the same principles in detecting and measuring enzyme activities, but only differs in the light wavelength from the UV spectroscopy. The MTT assay is a typical example of colorimetric assays. The MTT assay uses a tetrazolium dye as the substrate to assess cell metabolic activity. Generally, the number of viable cells or the vital level of cells can be reflected by the activity level of NAD(P)H-dependent cellular oxidoreductase enzymes. In vital cells, the enzymes reduce the tetrazolium dye MTT <3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide> to the formazan form, which has a purple color and can be easily used to quantify the enzyme activity. Other dyes can also be used for similar purposes, which includes XTT, MTS, and the WSTs, and often in conjunction with the electron acceptor PMS <1-methoxy phenazine methosulfate>. However, the solvent, reaction condition, and cell activity mechanisms have significant impacts on the results of the MTT or other assays. Therefore, a control test should always be performed by the side, and the results should be confirmed by another method.