Enzyme peak shift

A method of great value in biochemistry for the verification of peak identities is the enzymatic peak shift technique.5 The approach utilizes the specificity of enzyme reactions with a nucleotide or class of nucleotides. The technique is especially useful in the characterization of nucleotides of cell extracts, because not only is the identity of the reactant verified, but so is the identity of the product formed. With the enzyme peak shift method, one aliquot of the sample is analyzed while a second aliquot is incubated with an excess of an enzyme that catalyzes a specific reaction involving the compound of interest. After the enzyme is deactivated, the second aliquot is chromatographed. 

Enzymatic peak shift reactions are also useful for clarifying or unmasking a chromatogram. If one nucleotide is present in a large quantity, as for ATP in human erythrocytes, for example, it may hide the presence of a small quantity of another compound that has a similar retention time. Therefore, if an enzyme is added that converts the large peak into a product 

Photodiode array (PDA) detectors are becoming more popular as their ability to use both retention times and UV spectra to aid in peak identification becomes more widely recognized. With most PDA software packages, libraries of standard compounds can be created by the analyst. When samples are run, the spectra and/or the run times of the peaks in the sample can be compared with those stored in the libraries. Because the shapes of UV spectra are dependent on the experimental conditions under which they are obtained, however, it is unlikely that standard libraries will become commercially available. 

An example of the use of photodiode array detectors for determining peak homogeneity is in the analysis of /3-carotene,8 where an impurity in the all-trans isomer was positively identified as the cis isomer.9 Since the U.S. Federal Register began to require listing of the presence and concentration of various vitamins on the labels of foods, there has been increased activity in the measurement of these vitamins. /3-Carotene is one such vitamin, and research links the presence of /3-carotene to a reduction in the occurrence of some degenerative deseases such as cardiovascular diseases and cancer. 

As with PDA detection and the construction of UV libraries, computerized data systems are used for data collection and processing with mass spectral detection. Unlike UV spectra, however, mass spectra are much less dependent on experimental conditions. Thus, it is possible to purchase commercial libraries of mass spectra for spectral matching. Mass spectral data systems range from systems that record the spectra and produce conventional relative abundance bar charts for subsequent analysis to those 

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There are a variety of techniques currently in use to aid in the identification of sample components. Most techniques, such as spiking and the enzyme peak shift method, are used to confirm the identities of components thought to be in the sample. If, however, the identity of the sample is truly unknown, a combination of techniques is needed to provide a unique fingerprint. ... 

There are a variety of techniques available to aid in the identification of sample components matching retention times, standard addition, internal standard, isotopic labeling, enzyme peak shift, and UV and mass spectral libraries. 

LC-separation of low molecular-weight constituents of nucleic acids and intact nucleic acids was reviewed by Zadrazil [358,359], Brown [360] described an enzyme peak shift method verifying peak identities of nucleotides, Singhal [361] reviewed separation and analysis of nucleic acids and their constituents by ion-exclusion and ion exchange column chromatography, and Brown [31,362] summarized the latest developments and state-of-art in HPLC of nucleic acid constituents. Plunkett [363] dealt with the use of HPLC in research of purine nucleoside analogs. 

The enzymic peak-shift technique utilizes the specificity of enzyme reactions with a nucleotide or a class of nucleotides. It has proved useful not only in the identification of peaks but also in the removal of a peak so that concentrations of compounds represented by smaller peaks that are hidden by larger peaks can be measured. 

The chemical and spectroscopic properties of the cofactor F-430 have been reviewed [98,99], The structure of the macrocycle (Figure 4) was elucidated by x-ray crystallography and NMR spectroscopy [100], The free cofactor, which is present in substantial amounts in the cells, has an absorption maximum at 430 nm, hence its name. In the enzyme, the absorption maximum is blue shifted to 420 nm. The pentamethyl ester of F-430 is soluble in organic solvents and can be reduced to the Ni(I) state under aprotic conditions, resulting in an absorption peak shift to 382 nm [101], or can be oxidized to the Ni(III) state, giving an absorption peak at 368 nm [102],... 

Enzymatic or chemical peak-shift technique (use of a specific chemical or enzymic reaction to remove selected components of the mixture)... 

Electrochemical communication between electrode-bound enzyme and an electrode was confirmed by such electrochemical characterizations as differential pulse voltammetxy. As shown in Fig. 11, reversible electron transfer of molecularly interfaced FDH was confirmed by differential pulse voltammetry. The electrochemical characteristics of the polypyrrole interfaced FDH electrode were compared with those of the FDH electrode. The important difference between the electrochemical activities of these two electrodes is as follows by the employment of a conductive PP interface, the redox potential of FDH shifted slightly as compared to the redox potential of PQQ, which prosthetic group of FDH and the electrode shuttling between the prosthetic group of FDH and the electrode through the PP interface. In addition, the anodic and cathodic peak shapes and peak currents of PP/FDH/Pt electrode were identical, which suggests reversibility of the electron transport process. 

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