Enzyme assay sampling

Figure 9.77 HPLC chromatogram of a typical enzyme assay sample with NANA and CTP as substrates. Peaks A, CMP B, CMP-NANA. (From Petrie and Korytnyk, 1983.)...

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Fig. 2) and b) discrete sample processing using small test tubes, in order to ensure the physical separation of each enzyme assay (i.e., the LKB-8600) (Table I). 

Subsequent to a further washing step, to remove any unbound antibody-enzyme conjugate, the activity of the enzyme retained is quantified by a straightforward enzyme assay. The activity recorded is proportional to the quantity of antigen present in the sample assayed. A series of standard antigen concentrations may be assayed to allow construction of a standard curve. The standard curve facilitates calculation of antigen quantities present in unknown samples. 

Figure 8.13 Typical reaction trace of a coupled enzyme assay. The indicator reaction in many coupled assays will often show a demonstrable change after the addition of the sample but before the addition of the substrate for the test enzyme. This blank reaction may be due to the presence of endogenous substrates in the sample and its rate (B) must be measured in order to be able to calculate the activity of the test enzyme (T —B) from the total rate of reaction (7) which results from adding the substrate.

Variations in the substrate specificity of enzymes derived from different sources does occur and cross-reactivity should always be checked when developing an enzymic assay. This includes an investigation of the interference from a variety of substances that may be present in the sample in addition to studies on amino acid specificity. 

All assays performed on digester samples were conducted in 100 mM Tris buffer pH 7.5 with substrate incubations at 37 C. Glucose-releasing assays used the same Tris buffer with 0.5% sodium azide added. Colorimetric products from enzyme assays were detected and recorded using a Milton-Roy model 601 spectrophotometer equipped with sipper and data printer. 

Preparation and analysis of SDS extracts from digester sludge. The particulates from a 30-ml sample were removed by centrifugation (15,00Qg) at 4 C for 20 min. The particulates were washed three times with 100 mM Tris buffer pH 7.0 and resuspended in 15 ml of buffer. The extraction procedure consisted of agitating the sample with a Fisher model 346 rotator at 25 C in the presence of 0.1% SDS for 1 h. The particulate material was then removed by centrifugation at 15,000 g at 4 C for 20 min, and the supernatant was used to perform the enzyme assays. 

In 1985, Ruzicka and Hansen established the principles behind flow injection optosensing [13-15], which has subsequently been used for making reaction-rate measurements [16], pH measurements by means of immobilized indicators [17,18], enzyme assays [19], solid-phase analyte preconcentration by sorbent extraction [20] and even anion determinations by catalysed reduction of a solid phase [21] —all these applications are discussed in Chapters 3 and 4. Incorporation of a gas-diffusion membrane in this type of sensor results in substantially improved sensitivity (through preconcentration) and selectivity (through removal of non-volatile interferents). The first model sensor of this type was developed for the determination of ammonium [13] and later refined by Hansen et al. [22,23] for successful application to clinical samples. 

PTPS activity measurements are not available in external control programs and can only be compared by exchanging results from other laboratories (for examples see www.bh4.org). Control biopterin (and neopterin) samples are commercially available from Dr. Schircks Laboratories, Jona, Switzerland (www.schircks.com). We recommend using internal controls (e.g., normal control fibroblasts in each enzyme assay and standard biopterin for HPLC analysis see section 6.1.3.1, subheading Instrumentation ). 

To determine the mutagenic potential of nonaqueous liquids as measured by the Ames SaZmoneZ/a/mammalian-enzyme assay, the following protocol is recommended for the sample preparation. In step 1, the desiccator assay is performed on the neat material. The desiccator assay allows the detection of volatile mutagens (such as chlorinated solvents) that are often missed in the plate incorporation and pre-in-cubation assays (16, 17). In addition, a suspension of the neat material (20 mg/mL) is prepared by ultrasonication (5 min at room temperature) in high-purity DMSO (18, 19) and tested in the normal plate incorporation assay as well as in a pre-incubation Ames assay (20). The pre-in-cubation assay allows the detection of certain mutagens, such as dimethylnitrosamine, that require additional time for activation by mammalian or bacterial enzymes. A positive response in any of these three assays indicates the presence of mutagenic components, and the evaluation process is completed. 

G. K. Shoemaker, J. Lorieau, L. H. Lau, C. S. Gillmor, and M. M. Palcic, Multiple Sampling in Single-Cell Enzyme Assays, Anal. Chem. 2005, 77, 3132 X. Sun and W. Jin, Catalysis-Electrochemical Determination of Zeptomol Enzyme and Its Application for Single-Cell Analysis, Anal. Chem. 2003, 75, 6050. 

The goal of most enzyme assays is to quantitatively measure the amount of enzyme activity (catalytic activity) present in a sample. Thus, assay results are typically reported in activity units. A unit of activity may be defined in... 

The PGase extraction scheme outlined below (see Support Protocol) is based on the properties of the enzyme from tomato (Pressey, 1986). Enzymes from other sources will probably require different extraction conditions. A more detailed discussion of common approaches to enzyme extraction is included (see Background Information, discussion of samples for pectic enzyme assays). 

Fumaric acid has been found to be a good marker to detect the addition of D,L-malic acid to apple juice (Junge Spandinger, 1982). With the advent of the enzymic assay procedure for D-malic acid the method fell out of use. However, in 1995, a number of samples of apple juice in Germany were found to contain elevated levels of fumaric acid, which was attributed to the addition of L-malic acid. 

The tube is then decanted, washed twice or more with buffer and then an enzyme assay solution is added and the color development or change in absorbance is measured. The amount of enzyme remaining bound to the antibody will be a function of the amount of antigen present in the juice sample and, thus, the color development can be used to calculate the amount of juice antigen. 

Enzyme samples were added to test tubes and heated at a controlled temperature before conducting a standard activity assay. Samples were incubated either in boiling water for 15 min, or in a constant-temperature bath at 65, 85, or 105°C for 15 min. Samples were then cooled to room temperature and tested for activity at 65°C using the standard assay outlined above. To determine whether the substrate offered protection against thermoinactivation, another set of trials was conducted in which 0.5 g of corn was added to the enzyme during the preincubation period. The enzyme was then cooled and assayed for activity at 65°C, as outlined above. In these experiments, samples were incubated in a silicone oil bath at either 85,105, or 125°C. 

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