Photometric assays

For kinetic investigations and for activity measurements, either photometric assays or - because of the higher complexity of the reactants converted by biocatalysts - HPEC methods can often be used. Here the ionic liquid itself or impurities may interfere with the analytical method. 

Situation and Design A photometric assay for an aromatic compound prescribes the following steps ... 

Fungal cutinase catalyzes hydrolysis of model substrates and in particular p-nitrophenyl esters of short chain fatty acids, providing a convenient spectro-photometric assay for this enzyme activity [101,102,116]. Hydrolysis of model esters by this cutinase showed the high degree of preference of this enzyme for primary alcohol ester hydrolysis. Wax esters and methyl esters of fatty acids were hydrolyzed at low rates. Alkane-2-ol esters were hydrolyzed much more slowly than wax esters and esters of mid-chain secondary alcohols were not... 

John RA. 1993. Photometric assays. Enzyme assays. A practical approach. Eisenthal R, Danson MJ, editors. Oxford Oxford University Press pp. 59-92. 

John RA (1992) Photometric assays. In Eisenthal R, Danson MJ (eds) Enzyme assays a practical approach. Oxford University Press, p 59... 

In a photometric assay NADP(H)-dependent LBADH (see above) [9] and NAD(H) -dependent Candida parapsilosis carbonyl reductase (CPCR) [40] were identified as suitable catalysts accepting a broad range of ynones as substrates. Both enzymes catalyze the reduction of various aryl alkynones 21 with high enantioselectivity and efficiency (Scheme 2.2.7.13) [41]. 

The cross-linked aggregates of MeHNL also showed themselves to be highly active and robust catalysts. Optimized procedures give MeHNL CLEAs with activity recoveries up to 93% measured by a synthetic assay [75]. As observed earlier for PaHNL CLEAs [74], this result is in contrast with the photometric assay, indicating that a fast assay severely underestimates the recovery of initial activity because of rate-limiting diffusion [75]. 

The Sheldon group [87] prepared aquagels of different HNLs and compared them in the synthesis reaction of different cyanohydrins with the CLEAs and the free enzymes. The activity recovery for the aquagels and CLEAs measured by a photometric assay were quite low. Using the same loadings, the aquagels turned out to be much faster than the free enzyme. This confirms the underestimation of the recovery of activity by fast assays due to diffusion problems, as reported earlier [74, 75]. The stability and the catalytic performance of the immobilized HNLs are strongly influenced by the solvent, the immobilization method, and the enzyme source. 

The early studies of FDPase activity employed an assay based on the release of inorganic phosphate. More recently, a coupled spectro-photometric assay was introduced in which the fructose 6-phosphate formed is converted to glucose 6-phosphate, which is allowed to reduce TPN ... 

Photometric Assay of Color Developed by a Chemical Reaction... 

Kreft J. U. and Schink B. (1997) Specificity of O-demethyla-tion in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured by a coupled photometric assay. Arch. Microbiol. 167, 363—368. 

Another example of on-line monitoring of enzyme activities was given by Kunnecke et al. [88], when a FIA-system was used for the determination of enzyme activities during protein purification by fast protein liquid chromatography (FPLC). Photometric assays for four different oxidases were established in a FIA-system extending the linear range by the so-called zone sampling method. The FIA-device was coupled to the FPLC unit behind a... 

Gilis et al. [117] developed biosensors for the determination of L-alanine and its precursor pyruvate. They adapted the principle of a photometric assay, which was based on alanine dehydrogenase (Eq. (11.6))... 

The electrochemical detection utilized the re-oxidation of hexacyano-ferrate(II) on a platinum electrode. For pyruvate determination this assay was extended to a 3-enzyme system by the addition of glutamate p5u-uvate transaminase, which produces alanine from pyruvate. All enz5unes were used in solution in a reaction chamber of approximately 2 pi directly in front of the electrode. The cofactor NAD" " was coupled to dextran with a molecular weight of 40,000 to avoid its replacement for each assay. As the sensor responded to L-alanine and pyruvate again a differential measurement was required when a sample contained both compounds. It was applied to off-line monitoring of a cultivation of S. cerevisiae and data showed good correlation to the photometric assays. 

C, Photometric assay for an alkaline phosphatase label using a cascade detection reaction. p-iodonitrotetrazolium violet. 

When the mean bias is considered, one should distinguish between specific (systematic) sample-related interference (e.g., the influence of hemolysis on a photometric assay) in which a clear concentration dependent effect is present and general nonspecificity of the assay. The former can be handled appropriately, either by systematic corrections or by setting limits for allowed degrees of hemolysis. ... 

Atomic absorption spectrometry with flame (AA-F) or electrothermal atomization furnace (AA-ETA), inductively coupled plasma-emission spectroscopy (ICP-ES), inductively coupled plasma-mass spectrometry (ICP-MS), and high-performance liquid chromatography-mass spectrometry (LC-MS) are state-of-the-art analytical techniques used to measure metals in biological fluids. They are specific and sensitive and provide the cfinical laboratory with the capability to measure a broad array of metals at clinically significant concentrations. For example, ICP-MS is used to measure several metals simultaneously. Photometric assays are also available but require large volumes of sample and have limited analytical performance. Spot tests are also... 

A suitable photometric assay is available for the assay of cycloserine materials that have a potency of 0.02 rag/gm or more. 

N. Corbin, T. Hugli, and H. Miiller-Eberhard. Serum caiboxypeptidase B a spectro-photometric assay using protamine as a substrate. Anal. Biochem. 75 41-51 (1976). 

As discussed in the introduction, any enzymatic activity that is direcdy (or indirectly) linked to oxygen consumption could be measured by using the Hb02 method. However, it is not our purpose to shift all analytical procedures using manometric or polarographic techniques to spectro-photometric assays merely because it is more sensitive. Therefore, in the following sections we will discuss those applications where HbC>2 method could offer obvious advantages over other procedures. 

In agreement with the very low rate of reaction, HEL was unable to inhibit the proteolytic activity of PAP in a standard photometric assay. However, it is an important finding that the reaction can be observed and that the characteristics with respect to regio- and stereoselectivity are the same as observed with low molecular weight thiols. To our knowledge, this is the first instance in which reaction of an STL with the SH group of a protein was directly observed. 

Enzymes, such as glucoamylase and 3-glucosidase, which differ in their secondary and tertiary stmctures, were adsorbed on SPEB. ° The activity of the enzymes was analyzed in terms of the Michaelis-Menten parameters Km and fecat. This study demonstrated that the activity of the enzymes was preserved despite the dense packing of proteins within the bmsh (Figure 31). Recently, it was shown that ITC is a useful tool to study the activity of immobilized enzymes. This technique allows studies of the enzymatic activity in turbid media, such as in concentrated suspensions of the SPEB. Moreover, ITC does not demand chromophores or fluorophores as photometric assays do. Thus, this method is very versatile because unlabeled substrates can be used. The results from ITC were in agreement with a typical photometric study, and showed that the activity of (1-glucosidase adsorbed on the SPEB is retained. 

Takeguchi C and Sih CJ. A rapid photometric assay for prostaglandin synthetase application to the study in... 

Loiseau, P. M. Bories, C. Sanon, A. A new photometric assay with bromocresol purple for testing in vitro antitrichomonal activity in aerobic environment. Arzneim.-Foracfi. 1999, 49, 51-55. 

Kiianitsa, K., Solinger, J. A., and Heyer, W. D. (2003). NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 321, 266-271. 

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