Activity assay procedure

Both nicotinic acid and nicotinamide have been assayed by chemical and biological methods. Owing to the fact that niacin is found in many different forms in nature, it is important to indicate the specific analyte in question. For example, if biological assay procedures are used, it is necessary to indicate whether the analysis is to determine the quantity of nicotinic acid or if niacin activity is the desired result of the analysis. If nicotinic acid is desired, then a method specific for nicotinic acid should be used. If quantitation of niacin activity is the desired outcome, then all compounds (bound and unbound) which behave like niacin will assay biologically for this substance (1). 

The guideline states that the objective of validation is to demonstrate that an analytical method is fit for its purpose and summarizes the characteristics required of tests for identification, control of impurities and assay procedures (Table 13-2). As such, it applies to chiral drug substances as to any other active ingredients. Requirements for other analytical procedures may be added in due course. 

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

Microbiological assay procedures for nalidixic acid have also been used for biological samples. Since nalidixic and hydroxynalidixic acids have the same order of antibacterial activity in-vitro, then cannot be determined separately. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

An assay procedure based on the Straub tail reaction in mice has been shown to be capable of ranking the activities of both agonists and antagonists, results which are consistent with previous estimates [241]. 

A spectrophotometric assay procedure was used to measure the lipoxygenase isoenzyme activities of fresh and stored soya containing bread improver pastes and powders (77). 

All assays should be performed under reduced or F40 gold fluorescence lighting to minimize the potential for photodecomposition or activation. Assays are run in triplicate to determine percent inhibition. The tests are repeated at least once with a freshly prepared sample. If there is greater than 15% coefflciency of variation, the samples are run at least one additional time. When the reaction mixture is incubated within the plate reader, readings are taken immediately and at set times throughout the prescribed incubation period as established by the microsome supplier. For assays incubated outside of the plate reader, reactions were stopped in accordance with the product test procedure. 

The conditions used in an enzyme assay depend on what is to be accomplished by the assay. There are two primary applications of an enzyme assay procedure. First, it may be used to measure the concentration of active enzyme in a preparation. In this circumstance, the measured rate of the enzyme-catalyzed reaction must be proportional to the concentration of enzyme stated in more kinetic terms, there must be a linear relationship between initial rate and enzyme concentration (the reaction is first order in enzyme concentration). To achieve this, certain conditions must be met (1) the concentrations of substrate(s), cofactors, and other requirements must be in excess (2) the reaction mixture must not contain inhibitors of the enzyme and (3) all environmental factors such as pH, temperature, and ionic strength should be controlled. Under these conditions, a plot of enzyme activity (p-rnole product formed/minute) vs. enzyme concentration is a straight line and can be used to estimate the concentration of active enzyme in solution. 

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. 

Workers in several laboratories have noted that the activity of urease appears to increase upon standing at room temperature. Until this is understood assay procedures cannot be assumed to yield precise values. 

Pyrrolidone carboxylyl peptidase activity has been followed using pyrrolidone carboxylyl L-alanine (or other pyrrolidone carboxylyl-amino acids) as substrate by following the amount of amino acid released as determined by the ninhydrin method (137). In another assay procedure the release of -naphthyl amine from L-pyrrolidone carboxylic-/ -naphthylamide (135) is determined by the diazotization procedure of Bratton and Marshall (138) as modified by Goldberg and Rutenberg (139). 

Carbon-14 is usually produced in the form of barium carbonate which is therefore the starting point for the synthesis of 14C-labelled compounds fortunately, many such compounds at high specific activity are now available commercially. The isotope emits only /8-rays the maximum energy of which is about 0.15 MeV the rays are therefore only feebly penetrating and for this reason the radiation hazard in work with carbon-14 is small but special methods of assay are required to achieve great sensitivity. The most important of the assay procedures are ... 

Enzyme Activity Assays. The various enzyme activity assays used in the purification procedures are summarized in Table II. 

Poly-C-SpecificRibonuclease (P-RNase) (EC3.1.27.5). Warshawand Fournier (W3) showed that an increase in plasma enzyme activity of pancreatic P-RNase in patients with AP may indicate necrotic lesions, and is one of the few direct markers of pancreatic tissue injury (Nl, W4). Due to the time-consuming and cumbersome nature of the P-RNase assay procedure and the development of effective visualization techniques providing direct information on the structure of the inflamed pancreas, the diagnostic utility of the P-RNase assay has not been extensively studied (Table 3). 

This method of identifying the structure of the most active component of combinatorial libraries of mixtures is known as deconvolution (see section 6.5). It depends on both the mixtures containing the active compound giving a positive result for the assay procedure. It is not possible to identify the active structure if one of the sets of mixtures gives a negative result. Furthermore, complications arise if more than one mixture is found to be active. In this case all the possible structures have to be synthesized and tested separately. However, it is generally found that the activities of the library mixtures are usually higher than those exhibited by the individual compounds responsible for activity after they have been isolated from the mixture. 

Glutathione-peroxidase activity was measured spectrophoto-metrically at 340nm by an enzyme coupled assay procedure of Paglia and Valentine (28) as modified by Reddy, et al. (26). A molar extinction coefficient of 6.2 x 103cm 1 was used in calculations. 

The reaction rale that is measured depends on a number of experimental conditions such as temperature, pH, ionic strength, and the presence or absence of inhibitors or activators. In actual assays one usually chooses conditions ensuring iwwiwti iwriMB iste, It is only under the conditions specified in the- prescribed assay procedure that enzyme units are defined. Whenever different conditions are used, the rate measured does not have a well-defined relationship to the potency... 

Activity is measured by the procedure of Shugar.21 To 2.9-ml cuvettes (1 cm path length), diluted lysozyme (ranging from 0.1 to 0.5 nM) and antibody (ranging from 0.013 to 50 nM) are added to 66 mM potassium phosphate buffer, pH 6.24, and 0.1% bovine serum albumin (BSA) (w/v) to a volume of 900 fil. The solutions are kept at 25° for 1 hr to allow the lysozyme-antibody complexes to come to equilibrium. The activity assays are initiated by the addition of 100 pi Micrococcus lysodeikticus (Sigma Chemical Company) cell walls (2 mg/ml in 66 mM potassium phosphate, pH 6.24) to a final A450 of 0.8 -1.0. Cuvettes are wrapped with Parafilm to prevent evaporation, inverted several times to mix, and placed in a Perkin-Elmer, Norwalk, CT) Lambda 4B spectrophotometer. Reactions are monitored by the decrease in A450 for 70 min with a data point collected every minute. 

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