Structural Biochemistry/Proteins/Measuring Enzymatic Activity Using Spectroscopy
What is an Enzyme?
Enzymes are powerful biological catalyst. Catalysts speed up a reaction but are not consumed in the reaction. Enzymes are thus essential for such bodily functions as digestion because otherwise, these reactions would occur at too high of temperatures for the body to handle. The catalysis process takes place at the active site. Enzymes are extremely selective with their reactants, or substrates, and the type of chemical reactions they are involved with. This is due to similar shape, charge, and characteristics between the enzymes and the substrates. Bringing together the enzymes and the substrates is called enzyme-substrate (ES) complexes.
It has been observed through experimental analysis that enzymatic transition states often use kinetic isotope effects to understand the bonding differences between reactants and the intermediates found in the transition states. Kinetic isotope effects refer to the ratio of the rates of reaction of two different isotopically labeled molecules in a given reaction. In addition to transition states, enzymes also play an important role as common pharmaceutical targets since many drugs act as enzyme inhibitors. The most common feature of enzymes is that they are able to catalyze reactions and increase the reaction rates. They can also overcome larger single transition state energy barriers by breaking them down and creating multiple steps of smaller barriers. Reaction rates are also limited by conformational changes that occur in proteins as well as the rate at which reactant are released to yield product. In addition, the values of the kinetic isotope effects, often intrinsic, typically are the result of differences in the bond environments for atoms in the reactant state compared to the bond environments found in transition states.
Spectroscopy Measures Enzymatic Activity
The ability to determine enzymatic activity is extremely important to clinical chemistry because it allows for early diagnosis of various diseases and helps doctors determine the course of treatment for such diseases. The spectroscopic characteristics of enzymes and substrates change when they combine to form an ES complex. In order to measure this activity demonstrated by the enzyme, the following spectroscopic techniques are used: Fluorescence spectroscopy, UV/VIS Spectroscopy, Spectrophotometric Assays, and Infrared spectroscopy.
Fluorescence spectroscopy reveals the existence of ES complexes and what they are made of. In Fluorescence spectroscopy, a compound is exposed to UV-light which excites certain molecules and causes them to emit light at a lower wavelength, which is typically in the visible light range. This phenomenon in which the molecule's absorption of a photon at one wavelength leads to the emission of another photon from the same molecule at a longer wavelength is known as fluorescence. In this spectroscopic technique, the fluorescence of the substrate is measured and compared to the fluorescence of the product, and it is in the difference of these two measurements that enzymatic activity is measured.
A typical procedure for light-extinction measurements is as follows. At predetermined time intervals, 5-7 measurement points are collected by measuring the light-extinction of the enzyme sample, where a light-absorbing substance is either consumed or produced during the reaction. This can be measured using a photometer. The mean value and standard deviation of these measurement points are then found and plotted versus time. Then a regression curve is drawn through these points. The enzyme activity can then be found from the slope of the regression curve at a particular time
Many impurities found in fluorescent compounds, when exposed to light, interfere with the spectroscopy, making this technique more sensitive than other assays.
A method that is complimentary to fluorescence spectroscopy is ultraviolet-visible spectroscopy in that fluorescence spectroscopy deals with transitions from the excited state to the ground state and ultraviolet-visible spectroscopy deals with transitions from the ground state to the excited state. UV spectroscopy uses light in the UV region where molecules are most likely to undergo electronic transitions. What is meant by electronic transitions is that when a molecule absorbs UV energy, this causes the electrons to become excited, meaning they quickly and unstably move into a higher energy orbital. The instrument used in UV spectroscopy is a UV/VIS spectrophotometer. This device measures the transmittance of light through a sample. The equation used to calculate the transmittance is A=-log(%T) where A is the absorbance and T is I/Io where I is the intensity of light passing through the sample and Io is the initial intensity of the light, before it is transmitted through the sample. Once the absorbance is calculated, it can be plotted versus the wavelength giving a UV/VIS spectrum.
Spectrophotometric assays can track the course of a reaction by measuring how much light the assay absorbs. When the light is absorbed in the visible light region (400-750nm), the assay will actually change colors. This is called a colorimetric assay. An example of a colorimetric assay is the MTT. In the MTT assay, Yellow MTT(3-(4.5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) reduces to purple formazan in the mitochondria of cells, giving the reaction a purple color which allows for analysis of the enzyme. Some type of solution is used to dissolve the purple formazan product. A spectrophotometer is then used to measure the wavelength, which is usually between 500-600nm. The solvent that is used determines the maximum absorption wavelength.
Ultraviolet-visible spectroscopy is a commonly used spectrophotometric assay that examines photons in the UV-visible region. It is mainly used to determine the amount of a highly-conjugated organic compound or enzyme contained in a specific solution. The Beer-Lambert Law is used in Ultraviolet-visible spectroscopy to determine the concentration of the species that is absorbing the light. The Beer-Lambert Law states:
This equation shows the directly-proportional relationship between the solutions' concentration and the absorption of the solution, where A is the absorption of the solution and I refers to its concentration.
Another type of spectroscopy that can be used to obtain information about enzyme-substrate complexes is infrared spectroscopy. In enzyme-substrate complexes, there is well-organized binding modes, which is quantifiable using infrared methods. In analyzing infrared data, it is possible to identify binding modes and heterogeneity of ES complexes.
Detectors are needed to actually measure the enzymatic activity produced in the previously described processes. The following describes typical detectors used in industry.
UV/visible light detectors are the most common used in industry because they are versatile, have a wide dynamic range, have a high sensitivity, and they are not affected by temperature and flow variations.
Fixed-wavelengths detectors use lamps that emit light at certain wavelengths. To select a particular wavelength a cut-off filter can be used. Fixed-wavelength detectors are good because they don't produce a lot of noise, it doesn't cost very much to operate them, and operating them is relatively simple.
Variable Wavelength Detectors
Variable wavelength detectors are different from fixed-wavelength detectors in that they use a range of wavelengths, typically between 190-700 nm continuously instead of just one wavelength at a time. For wavelength selection in the variable wavelength detector a continuously adjustable monochromator is used. The lamp is typically deuterium or tungsten. Light from the lamp travels to mirrors that focus and steer the light into a diffraction grating, and the grating drive mechanisms are all part of the monochromator assembly. To monitor more than one wavelength at a time, the grating is rapidly adjusted between two different wavelengths.
One of the most recent developments for variable wavelength detectors is the Diode Array Detector (DAD). The DAD basically speeds up the entire process by setting multiple detectors next to each other on a silicon crystal and using a capacitor to convert light to electric charge. this makes it so that light from the grating can be detected much quicker than in a conventional spectrophotometer. The advantages to using a DAD over motor-driven monochromators are that they possess fewer moving parts and they are less likely to have irregular data at high flow rates.
Fluorescence detectors are used only when the compound can't be detected by the other methods and the compound must have fluorescence or can come to have fluorescence by reacting with a fluorescent compound. The intensity of light that is emitted is directly proportional to the power of exciting radiation, therefore fluorometric detection is much more sensitive than absorption. Usually the fluorescence detector is made up of a light source, a wavelength reflector and a single or dual-flow cell.
The light source is typically xenon, mercury arc, or quartz halogen. The wavelength reflector allows for the excitation spectra to be obtained for the compound. This information can be used for rapid method optimization and verification of separation quality. Lastly, the single and dual-flow cells take account the fluorescence of the mobile phase.
Refractive Index (RI) detectors
Refractive Index (RI) detectors measure the change in refractive indices in the reference and sample cells. The drawback to this method is that refracted indices between compounds can be very small, therefore the sensitivity of the RI detector is much smaller than for UV/VIS and fluorescence detectors. Another negative in using an RI detector is that the detector response is affected by the mobile phase composition, which means there is a lot of error in the final data collected. There are three types of RI detectors: deflection, fresnel, and interferometric, but the deflection type is the most commonly used.
In the deflection detector, a light beam passes between two parallel chambers in a glass prism, which acts as a reference. If the refractive index of the solution is equal to the refractive index of the reference, then the light beam is parallel to the incident beam. However, if the refractive indices are different, the beam is deflected and then measure by a differential photodiode.
Electrochemical detectors are most commonly used for biogenic amines because they are more sensitive and more selective than the previous methods. There are two types of electrochemical detectors: bulk property and solute property. The bulk property detectors are most commonly used and they measure the change in cell resistance. Solute property detectors monitor the change in potential or current as the solute passes through the cell. Solute is passed over an electrode which is held at a constant voltage. The current produced is proportional to solute concentration.
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