Enzyme colorimetric method analysis

The analytical chemistry of alcoholic beverages, of which the determination of ethanol is only one facet, continues to develop. However, several methods of analysis for ethanol are available and tradition, government legal requirements, and convenience, taken together or separately, usually govern the choice of method. These methods include (1) distillation and specific gravity measurement, (2) gas-liquid chromatography, (3) IR spectrometry, (4) headspace techniques, (5) enzyme methods, (6) chemical (colorimetric) methods, and (7) refractometry. These methods are now considered in turn. 

Barzana E, Klibanov AM, and Karel M (1989) A colorimetric method for the enzymic analysis of ethanol and formaldehyde vapors using solid alcohol oxidase. Analytical Biochemistry 182 109-115. 

A review on the subject of immobilized enzymes in biochemical analysis covers preparation and properties of immobilized enzymes assay methods using immobilized enzymes (spectrophotomatic assays, automated methods in biochemical analysis, enzyme electrodes, and colorimetric analyses using immobilized enzymes) and applications of immobilized enzymes in biochemical analysis. ... 

Biomedical analytical chemistry happens to be one of the latest disciplines which essentially embraces the principles and techniques of both analytical chemistry and biochemistry. It has often been known as clinical chemistry . This particular aspect of analytical chemistry has gained significant cognizance in the recent past by virtue of certain important techniques being included very much within its scope of analysis, namely colorimetric assays, enzymic assays, radioimmunoassays and automated methods of clinical analysis. 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. 

To improve the quality control process of gasohol and hydrated ethanol, an automated FIA system was developed using AOD and HRP enzymes, and addition of 4-aminophenazone and phenol. A colorimetric detection method was used in two different methods of analysis, with free (4) and immobilized enzymes. Both systems have shown good results when compared with established methods such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) (4,7). 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

The results of enzymatic determinations of ceruloplasmin are often expressed in arbitrary units, and the values judged in the light of a series of results obtained in normal subjects by the same method. Expression of the enzyme activity in milligrams of ceruloplasmin per unit volume of serum is also possible. The relation between oxidase activity and the amount of ceruloplasmin in serum can be determined by measuring in parallel samples of sera both the oxidase activity and the change of optical density at 610 mix before and after the addition of ascorbic acid or cyanide. On the basis of the known absorbancy index, the ceruloplasmin concentration can be calculated (see Section 2.2.1) and the relation between it and the enzyme activity determined. Alternatively, purified human ceruloplasmin can be used for standardization of the enzymatic method. The ceruloplasmin content of the purified preparation can be determined colorimetrically or, in the case of a highly purified preparation, by nitrogen analysis. Predetermined increments of ceruloplasmin can then be added to aliquots of a selected serum. It is convenient to select a serum with relatively low ceruloplasmin level to start with. Serum of a patient with Wilson s disease, some of whom have no measurable amount of enzyme activity, would be ideal for the purpose however, Walshe (W5) has recently found an inhibitor in these sera. 

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