Enzyme assay incubation times

Most enzymes show a bell-shaped pH-velocity profile and a characteristic pH at which their activity is maximal. Figure 5.13 shows V0 vs pH curves for three enzymes. Note that both the pH optimum and the form of the velocity profile vary with the enzyme. Such curves must be interpreted with caution, as they give no indication why the velocity declines above and below the pH optimum. The decline in rate may be due to the formation of improper forms of the enzyme or substrate (or both) or inactivation of the enzyme, or it may be due to a combination of these factors. The possibility of enzyme inactivation is frequently overlooked, although a pH stability curve is necessary for enzyme characterization. A pH stability curve is readily obtained by preincubating the enzyme at a specified pH for a period of time equal to the assay incubation time and then assaying activity at the optimum pH. 

Therefore, assay incubation times can be reduced from hours to minutes. The purified biomarkers can be detected via the enzyme-antibody conjugates without compromising sensitivity. Since a large bulk solid phase is not present during the immunospecific capture step, nonspecific binding is also minimized, which can reduce the assay background noise to improve the limit of detection. 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Several things may be done if the researcher has difficulty in detecting an enzyme activity of interest in a homogenate, or elsewhere. A more sensitive assay technique may be used, if one is available. The concentration of enzyme may be increased, as the rate of product formation is directly proportional to [E]. The incubation time for enzyme with substrate can be increased, although the caveats discussed in O Section 3.3.2 must be borne in mind. The reaction volume may also be increased, while maintaining concentrations of reactants constant this approach is particularly useful if product is separated and detected by chromatography, or if a column is used to separate radiolabeled substrate from product, because the increased amount of product formed in unit time will result in enhanced signal size. 

To study the effect of hydrogen peroxide, the lignin peroxidase was incubated in buffer, either pH 3.0 or 5.0, at the temperature of either 0°C or 10°C. Protein concentration was 30/i,g/ml and the concentration of H2O2 was 0.2 -11.6 mM. The incubation time varied between 0 and 295 min. After incubation 0.4 ml of the enzyme sample was pipetted directly into the activity assay mixture. 

In determining enzyme activities, it is usually assumed that at a fixed set of so-called saturating substrate concentrations a sufficiently accurate value of F, ax is obtained. Bisubstrate kinetic analyses of UDP-glucu-ronyltransferase [assayed with bilirubin (P5) and p-nitrophenol (V6), respectively] indicate that a true measure of the amount of enzyme can be obtained only by suitable extrapolation procedures. This restriction applies in particular to bilirubin (A2, HIO, T8) and other aglycons (M15, V6) because of substrate inhibition. UDP-glucuronic acid was inhibitory at concentrations only about 10-fold higher than the apparent Km value (HIO) this was most pronounced at relatively short incubation times. Mg was noninhibitory at concentrations equal to 20 times the apparent Km values (F3, HIO). 

Figure 2. Heat inactivation profile of the xylanase in the culture filtrate from Thermoascus aurantiacus. Xylanase enzyme was prepared as described by Tan et al. (74). Xylanase and endocellulase activities were determined after the enzyme was incubated for the specified time at 70°C. Aliquots were removed and assayed at 50°C by the method described by Yu et al. (75). Activities were expressed as a percentage of the control stored at 4°C.

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. 

Figure 6. pH-activity profile of cellulolytic enzyme activities from Thermoactinomyces sp. under assay conditions. (Q) CM-cellulase, incubation time 10 min (A) Avicelase, incubation time 20 min (O) /3-glu-cosidase, incubation time 30 min. 

Aqueous invertase solutions (soluble and immobilized enzyme) were incubated in acetate buffer (0.010 M, pH5.5) for 20,40,60,80, and 120 min at 30,37,40,45,50, and 55°C. After incubation at each specified time, both forms were assayed for residual activity according to the standard procedure (see Standard Assay for Measuring Invertase Activity). 

The enzyme was incubated for a given amount of time at different temperatures, after which the activity was measured by the standard assay at 30, 40, and 50°C using MCD as the substrate. GOx was used for in situ production of H202. 

Twenty percent (v/v) mycelium suspension was used to inoculate 500-mL conical flasks containing 15 g of corncob as carbon source and 22.5 mL of PPMKC medium (pH6.0) as optimized by Damaso et al. (6). After inoculation, the flasks were incubated in a stationary manner at 45°C for 6 d in a laboratory electric incubator. At each sampling time, the culture medium was vacuum filtered using filter paper (Whatman, no. 4, fast-flow rate), and the filtrate was used for further enzyme assays. During the cultivation, two or more flasks were sampled daily. 

In some cases the rate of enzyme inactivation can be quantified without an assay for enzyme activity. For example, inactivation of CYPs due to MIC formation can be directly quantified spectrophotometrically, which avoids the potential artifacts introduced by the measurement of catalytic activity. Microsomes, or purified enzymes, are incubated with a substrate and NADPH and monitored for MIC formation over time in a spectrophotometer. An example of MIC formation by diltiazem in human liver microsomes (HLMs) is shown in Figure 3 (36). The MIC exhibits an absorbance maximum between 448 nm and 456 nm when the heme iron is in the reduced state (17). Extinction coefficients of MIC are approximately 64mM/cm (79). Thus, MIC formation by diltiazem in the example is 59% of the total CYP, which would be consistent with inactivation of most of the CYP3A in the microsomes. 

The ELISA procedure for the analysis of parathion as described above requires nearly eight hours, although many samples can be simultaneously assayed. However, incubation times can be shortened to one-half hour, in most cases, resulting in only a 10% reduction in sensitivity. Also the polystyrene microtiter plates containing bound RSA-AP can be mass produced and stored in a freezer. Since the enzyme-linked antibody can be purchased, the limiting factor of the applicability of the ELISA procedure, as well as the RIA procedures, for other pesticides is the development of the antiserum to the pesticide. 

After incubation of the enzymes for various times at the indicated temperatures in the presence of 1 mM CaCh, remaining activity was measured by assaying as described for Table 12.2. Half-life was determined from semi-log plots of logio[remaining activity] versus time. 

If protease inhibitors should be identified and characterized, the assay should be tested for signal stability and endpoint linearity in the next step. The progress curves recorded for substrate concentrations near or below the KM value should be linear for up to 2 h. Such signal stability is a prerequisite to run the assay with a pre-incubation time of 1 h for enzyme and inhibitor. This long pre-incubation is recommended to ensure also that the IC50 values for slowly binding inhibitors are correctly determined. An additional hour for the incubation of enzyme, inhibitor and substrate after this pre-incubation is recommended. 

Figure 9.45 Chromatography of enzyme assay media. Peaks 1, aspartate 2, glutamate 3, asparagine 4, glutamine 5, Tris-HCl buffer. Elution profile of the assay medium incubated for (A) zero time and (fl) 30 minutes. (From Unnithan et al., 1984.)...

The standard assay contained 100 vaM Tris-HCl (pH 9), 100 mAf KG, 20 mM threonine and/or serine, and the enzyme. After incubation at 37°C for various times, the reaction was terminated by addition of 3 N HQ. The reaction mixture was centrifuged and 250 / L of the supernate was mixed with 250 /iL of o-phenylenediamine solution (10 mg/mL in 3 N HQ). The tubes were tightly capped and heated in a boiling water bath for 30 minutes. The derivatized keto acids were extracted with ethyl acetate. An aliquot of the ethyl acetate layer was dried under nitrogen. The residue was taken up in 1 mL of the running solvent for HPLC Assays were linear for 30 minutes and with amount of protein added. 

Figure 9.87 Elutions of the nicotinate phosphoribosyltransferase (N-PRTase) assay solution through a /xBondapak C18 column after various enzyme incubation times. The incubation mixture contained 5 mM MgCl2,100 y,M nicotinate, 75 tiM ATP, 30 /iM PRibPP, and 25 yu.L of 4 mg/mL N-PRTase in 50 mM Tris-HQ (pH 8). Elution conditions 5 yuL sample injection volumes, 0.7 mL/min flow rate, 25 m M (NH))P04 (pH 8) elution buffer, 25°C. (From Hanna and Sloan, 1980.)...

Enzyme assays were performed in 1.5 mL microcentrifuge tubes containing 165 /nL of incubation buffer (0.25 M Tris-HCl, 0.1 mM EDTA, pH 7.4), 50 fiL of 10 mM glutathione in incubation buffer, and 25 /nL of appropriately diluted enzyme. The reaction was initiated by addition of 10 /nL of benzo [a]pyrene-4,5-oxide in acetonitrile (0.875 mg/mL). The assay was stopped by addition of 0.25 mL of acetonitrile containing 300 /nM 2-methoxynaphthalene as an internal standard. The solution was kept overnight in the dark at 4°C, and then 500 /nL of distilled water was added. The mixture was centrifuged before analysis by HPLC. Production of both the glutathione and diol derivatives was linear with time and protein up to 15 minutes and 500 /ng/mL, respectively. 

In this technique, an aliquot of the sample under investigation is first chromatographed. A second aliquot of sample is incubated with the enzyme under the appropriate conditions of pH and temperature. After a suitable time period, the incubated mixture is chromatographed. The disappearance of the substrate peak and/or appearance of product peaks confirms the identity and purity of the chromatographic peak. In cases where specific enzymes may not be available for a certain substrate, less specific enzymes, such as phosphatases, can be used for the identification of classes of compounds. In addition, it is possible to use coupled enzyme assays to drive a reaction to completion and thus characterize a peak in the chromatogram. 

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