Fluorometric enzymatic methods

Two disparate translation methods are investigated for the measurement of sulfur dioxide. Both involve interaction with an aqueous solution. In the first, collected S(IV) is translated by the enzyme sulfite oxidase to which is then measured by an enzymatic fluorometric method. The method is susceptible to interference from i CWg) efforts to minimize this interference is discussed. The second method involves the translation of SO2 into elemental Hg by reaction with aqueous mercurous nitrate at an air/liquid interface held in the pores of hydrophobic membrane tubes. The liberated mercury is measured by a conductometric gold film sensor. Both methods exhibit detection limits of 100 pptv with response times under two minutes. Ambient air measurements for air parcels containing sub-ppbv levels of SO2 show good correlation between the two methods. 

Enzymatic Fluorometric Method I. There are several factors, such as enzyme concentration, substrate concentration, pH of buffer, and temperature, which can affect the kinetics of the enzyme catalyzed reaction. These factors should be optimized and carefully controlled In order to obtain the most sensitive and reproducible results. The results of the optimization studies are summarized In Table I. 

Table I. Optimal Concentrations of the Reagents Used in the Enzymatic Fluorometric Methods...

Enzymatic Fluorometric Method II concentration of formaldehyde dehydrogenase, diaphorase, NAD, resazurin and the pH of buffer were optimized. The results of the optimized parameters are also shown in Table I. The times required to obtain the steady state (of about 3 minutes) at different formaldehyde concentrations are shown in Figure 3. 

The slopes of the calibration plots, 60.87 fluorescence unit per Pg/ml for enzymatic fluorometric method I and 120 fluorescence unit per pg/ml for enzymatic fluorometric method II, show that method II is approximately twice as sensitive as method I. This is due to formation of the intensely fluorogenic resorufin in method II. The higher sensitivity and lower detection limit of the enzymatic fluorometric method II will have potential applications in air sampling of formaldehyde emissions since sampling time can be reduced. 

Figure 2. Calibration curve for the enzymatic fluorometric method...

Figure 4. Calibration plots for the enzymatic fluorometric method II in ppm and ppb levels.

We have developed two novel new enzymatic fluorometric methods for the trace analysis of formaldehyde. Due to their simplicity, sensitivity and specificity, these methods should find wide applications in the monitoring of formaldehyde released from wood products. As we stated above, enzymatic fluorometric method II does offer higher sensitivity and better detection limit over enzymatic fluorometric method I. However, method II requires two... 

Chapman J. and M. Zhou. 1999. Microplate-based fluorometric methods for the enzymatic determination of l-glutamate Application in measuring l-glutamate in food samples. Anal. Chim. Acta 402 47-57. 

The histamine content of the supernatants can be determined using the automated fluorometric analyzer (12). Other methods used include a radioisotopic-enzymatic assay (13) or an enzyme immunoassay (14). 

Riboflavin can be assayed by chemical, enzymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of iatense yeUow-green fluorescence with a maximum at 565 nm in. neutral aqueous solutions. The fluorometric determinations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the kradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

ASTM. 1994. Proposed test method for fluorometric determination of toxicity-induced enzymatic inhibition in Daphnia magna. ASTM 1994 Annual Book of Standards Vol. 11.04, E-47 Proposal P 235. American Society for Testing and Materials, West Conshohocken, PA, pp. 1717-1721. 

Two sensitive fluorometric enzymatic methods for the determination of formaldehyde release from wood products were described. These methods were developed using the enzyme formaldehyde dehydrogenase to catalyze the oxidation of formaldehyde to form formic acid and NADH in the presenc of oxidized nicotinamide adenine dinucleotide (NAD ). The increase in NADH, which is directly proportional to the concentration of formaldehyde, is measured fluorometrically at em ... 

Physicochemical assays, including spectrophotometric, fluorometric, chromatographic, enzymatic, immunological, and radiometric methods... 

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

Choline content of foods is usually determined by a colorimetric method or by microbiological assay. Recent assay techniques include fluorometric enzyme assay, enzymatic radioisotopic eissay, and gas chromatography. 

Optical detectors are by far the most commonly used of the analytical methods available for continuous monitoring. In fact, in around two-thirds of all FIA applications reported the use of this type of detector. However, several other types of detection methods are available for the determination of ions acetate and acetic acid involving, for instance, potentiometric, enzymatic, spectrophotometric, fluorometric, and chemiluminescence techniques. However, molecular absorption spectrophotometers continue to be the preferred choice in FIA, and in analytical chemistry in general, on account of their high versatility (Calatayud, 1996). Studies employing FIA and other methods for the determination of acetic acid and acetate ions are described in the following sections. 

This reaction was exploited by Tsukatani and Matsumoto (2000) in a stopped-flow FIA method. An immobilized D-malate dehydrogenase enzyme reactor was employed and the reduced enzymatic cofactor NADH that was formed was monitored fluorometrically (Xex = 340 nm = 460 nm). Due to the slow reaction rate, the flow was stopped with the sample in the reactor to increase reaction time. The intrinsic sample fluorescence was also assessed using a parallel blank reactor without immobilized enzyme. The method was validated through the analysis of red and white wine samples. The enzyme reactor stability was also evaluated and it was found that the sensitivity (evaluated as amplitude of response at a constant concentration of the analyte) gradually decreased to 60% within a week but then remained stable for a month. As D-malate cannot be present in naturally fermented wines (except for fraudulent addition), the interference of this primary substrate can be considered negligible. 

Alternative methods have been proposed based on spectrophotometric [13] and fluorometric [14] techniques. But undoubtedly, the most widespread methods for MSG quantification are electrochemical methods using enzymatic biosensors. The latter combine the advantages of the sensitivity of these techniques and the specificity of enzymatic reactions. 

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