Enzyme Assays Lysozyme Activity

Goven, A.J., S.C. Chen, L.C. Fitzpatrick, and B.J. Venables. 1994. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes enzyme assay for immunotoxicity of xenobiotics. Environ. Toxicol. Chem. 13 607-613. 

Enzymic Activity. Lysozyme activity was determined by following the rate of lysis of dried Micrococcus lysodeikticus cells according to the method of Shugar ( ). Assays were run at room temperature in O.IM phosphate buffer pH 7.0, with an enzyme concentration of about 0.05 mg/ml. A solution of native lysoz3mie at the same protein concentration was always assayed as standard, along with ozonized lysozymes. 

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

Fig. 1. Effect of HyHEL-10 on M. lysodeikticus lysis activity of chicken lysozyme at 0.1 nM concentration using the competitive inhibition enzyme assay described in the text (a) Raw data with varying concentrations of antibody and the substrate-only control, (b) Plot of antibody concentration versus decrease in absorbance at 450 nm per minute at 0.1 nAf enzyme (unconected for substrate-only control).

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

The rate of an enzyme-catalyzed reaction as a function of pH generally yields a bell-shaped curve. Some enzymes are very sensitive to small changes in pH (lysozyme), whereas others (POase) are relatively insensitive (within 1-2 pH units near their optima). Enzymes with similar activities but from different origin may have very different optima. APase from Escherichia coli is optimally active at a pH of about 8, whereas APase from calf intestine is most active around pH 10 and the activity of these enzymes decreases strongly outside their optima. Nevertheless, the E. coli enzyme is often assayed at the pH optimum of the intestinal enzyme. The optimum substrate concentrations may also be pH dependent for some enzymes (Chapter 10). 

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). 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Gonzalez, J. M., Sherr, B. P., and Sherr, E. B. (1993). Digestive enzyme activity as a quantitative measure of protistan grazing The acid lysozyme assay for bacterivory. Mar. Ecol. Prog. Ser. 100, 197-206. 

The use of optical rotatory dispersion in quantitative analysis has been applied to the microdetermination of lysozyme. A spin-label assay for lysozyme which is based on the enzymic hydrolysis of spin-labelled peptidoglycan has been described. Hydrolysis of this polymer by lysozyme results in sharpening of the e.s.r. spectrum. According to this method, human lysozyme is 3.5 times as active as hen egg-white lysozyme. The assay is suitable for measuring lysozyme levels in biological fluids. 

Based on these results, Dordick et al. [78] did another study to investigate how the sizes of the silica nanoparticles infiuence the structure and enzymatic activity of adsorbed lysozyme. The same CD technique was used to observe the protein structure, and calorimetric activity assays were used to monitor the enzyme activity after adsorption. The results from the two analysis techniques were then used to... 

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