The usual procedure for (colorimetric) assays is to prepare a set of standards and produce a plot of concentration versus absorbance called a calibration curve. This should be linear as long as the Beer–Lambert law applies. Absorbances of unknowns are then measured and their concentration interpolated from the linear region of the plot. It is important that one never extrapolates beyond the region for which an instrument has been calibrated, as this potentially introduces enormous errors.
To obtain good spectra, the maximum absorbance should be approximately 0.5, which corresponds to concentrations of about 50 μM (assuming ε = 10 000 dm 3 mol−1 cm−1 ).
Qualitative and Quantitative Analysis
The absorption spectrum is the plot of absorbance versus the wavelength (or the frequency). The wavelength of maximal absorption λ max is the wavelength for which a maximum is observed in the absorption spectrum and at which the sample possesses a corresponding molar extinction coefficient of
These wavelengths and molar extinction coefficients can allow identification of a given compound in a given solvent. However, such identification is rather limited and qualitative analysis is only possible for systems where appropriate features and parameters are known, such as identification of certain classes of compounds both in the pure state and in biological mixtures (e.g. protein-bound).
Most commonly, this type of spectroscopy is used for quantification of biological samples either directly or via colorimetric assays. In many cases, proteins can be quantified directly using their intrinsic chromophores, tyrosine and tryptophan. Protein spectra are acquired by scanning from 500 to 210 nm. The characteristic features in a protein spectrum are a band at 278/280 nm and another at 190 nm (Figure 1). The region from 500 to 300 nm provides valuable information about the presence of any prosthetic groups or coenzymes. Protein quantification by single wavelength measurements at 260 and 280 nm only should be avoided, as the presence of larger aggregates (contaminants or protein aggregates) gives rise to considerable Rayleigh scatter that needs to be corrected for (Figure 1).
Fig1. The presence of larger aggregates in biological samples gives rise to Rayleigh scatter visible by a considerable slope in the region from 500 to 350 nm. The dashed line shows the correction to be applied to spectra with Rayleigh scatter, which increases with λ −4 . Practically, linear extrapolation of the region from 500 to 350 nm is often performed to correct for the scatter. The corrected absorbance is indicated by the double arrow.
Difference Spectra
The main advantage of difference spectroscopy is its capacity to detect small absorbance changes in systems with high background absorbance. Difference spectra can be obtained in two ways: either by subtraction of one absolute absorption spectrum from another, or by placing one sample in the reference cuvette and another in the test cuvette. Difference spectra have three distinct features as compared to absolute spectra:
1. Difference spectra may contain negative absorbance values
2. Absorption maxima and minima may be displaced and the extinction coefficients are different from those in peaks of absolute spectra
3. There are points of zero absorbance, usually accompanied by a change of sign of the absorbance values. These points are observed at wavelengths where both species of related molecules exhibit identical absorbances (isosbestic points), and which may be used for checking for the presence of interfering substances.
Common applications for difference UV spectroscopy include the determination of the number of aromatic amino acids exposed to solvent, detection of conformational changes occurring in proteins, detection of aromatic amino acids in the active sites of enzymes, and monitoring of reactions involving ‘catalytic’ chromophores (prosthetic groups, coenzymes).
Spectrophotometric and Colorimetric Assays
For biochemical assays testing for time- or concentration-dependent responses of systems, an appropriate readout is required that is coupled to the progress of the reaction (reaction coordinate). Therefore, the biophysical parameter being monitored (readout) needs to be coupled to the biochemical parameter under investigation. Frequently, the monitored parameter is the absorbance of a system at a given wavelength, which is monitored throughout the course of the experiment. Preferably, one should try to monitor the changing species directly (e.g. protein absorption, starting product or generated product of a reaction), but in many cases this is not possible and a secondary reaction has to be used to generate an appropriate signal for monitoring. A common application of the latter approach is the determination of protein concentration by Lowry or Bradford assays, where a secondary reaction is used to colour the protein. The more intense the colour, the more protein is present. These assays are called colorimetric assays and a number of those commonly used are listed in Table 1 .
Table1. Common colorimetric and UV/vis absorption assays
Table1. (cont.)
