Enzymatic analysis coupled measurements

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

Chemiluminescence reactions are currently exploited mainly either for analyte concentration measurements or for immunoanalysis and nucleic acid detection. In the latter case, a compound involved in the light emitting reaction is used as a label for immunoassays or for nucleic acid probes. In the former case, the analyte of interest directly participates in a chemiluminescence reaction or undergoes a chemical or an enzymatic transformation in such a way that one of the reaction products is a coreactant of a chemiluminescence reaction. In this respect, chemiluminescent systems that require H2O2 for the light emission are of particular interest in biochemical analysis. Hydrogen peroxide is in fact a product of several enzymatic reactions, which can be then coupled to a chemiluminescent detection. 

With the advent of very sensitive ionization techniques such as matrix assisted laser desorption (MALDI) coupled with time-of-flight (TOF) mass analysis, measurement of the intact mass of peptides at sub-pmol levels has become a reality (2) of which we have taken advantage for the systematic screening of HPLC fractions. Partial sequence information can be obtained by carrying out enzymatic hydrolysis with exoproteinases (e.g. carboxypeptidases and aminopeptidases) (3, 4). More recently, MALDI has been used to measure metastable decomposition occurring in the first field free region of a reflectron TOF instrument (referred to as post source decay (PSD)) with only marginally more sample (5-7). 

For analytical purposes cholesterol oxidase has been immobilized on various carriers (Table 6). Electrochemical, optical, and calorimetric indication have been used as detection methods. Combination of a thermistor-coupled flow-through system with immobilized COD permitted the measurement of 0.03-0.15 mmolA cholesterol (Mattiasson et al., 1976). Ogren et al. (1980) described an immobilized COD reactor for the analysis of steroid fractions obtained by high pressure liquid chromatography. The UV absorption at 240 nm of enzymatically formed cholestenone was used as the measuring signal. Linearity was found between 10 and 80 pmol/1. 

The number of substances that can be measured by monoenzymatic approaches in electrochemical biosensors is limited, because in the majority of biocatalytic reactions electrochemically active compounds are not involved. To form readily detectable species, different enzymatic reactions have to be coupled, as is already routine in wet biochemical analysis [13]. This coupling can be accomplished in ways analogous to those present in a living cell. Here, nature provides us a variety of ways of regulating metabolic pathways. Thus, like in nature, catalytic activities of different enzymes can be combined in biosensors either in sequence, competing pathways, or in cycles (Table 4). 

The oxidized and reduced forms of the pyridine cosubstrate are readily distinguished by absorbance readings at 340 nm (Fig. 2.5). Therefore, whenever possible, enzymatic reactions which are difficult to measure directly are coupled with an NAD(P)-dependent indicator reaction (cf. 2.6.1.1) for food analysis. 

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