Lysozyme assay

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. 

Figure 8.16 Fluorimetric assay. Lysozyme assay system. Assay is performed under mildly acidic conditions but appearance of charged fluorophore requires pH- quenching to pH 9.

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. 

FRET to occur, and thus no FI emission is observed. The specificity of this assay was also examined for mixed samples. The mixed lysozyme samples were prepared in fetal bovine serum (FBS), human saliva and human urine. It was found that FAM emission was still visible upon addition of each mixed sample, implying that this assay has a great potential for the detection of real biological samples. This study illuminates that introduction of specific aptamer/protein interaction as the recognition event, and utilization of FRET as the signal transduction channel, is an effective way to develop CPE-based protein sensors with good specificity. 

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). 

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. 

Figure 1. Inactivation of lysozyme by ozone. Volumes of 4.8 ml of O.IM buffer containing 2.814 mg of lysozyme were exposed to a gas stream of ozone in oxygen (1.8 nmole/min ozone). Aliquots were removed for enzyme assay as described in Materials and Methods.

Figure 5. Separation of ozonized lysozyme CNBr fragments. The ozonized lysozyme was treated with CNBr as described in the Materials and Methods. The peptide mixture was applied to a column of Sephadex G-25 (3 X 100 cm) and eluted with 0.2N HAc at a flow rate of 0.5 ml/min. Fractions were assayed by measuring UV absorbance on ninhydrin color after alkaline hydrolysis.

Lysozyme is an enzyme that hydrolyzes some bacterial cell-walls, the bacterium used for the assay being Micrococcus lysodeikticus. Lysozyme is found in a wide variety of species and locations, including bacteriophages, blood, egg white, gastric secretions, milk, nasal mucus, papaya, sputum, and tears. The outstanding achievement in this field has been the elucidation of the crystal structures of some of the lysozyme-substrate complexes. 

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). 

Biophysical characterization showed that a single HRP II protein bound 17 molecules of heme [35]. In an in vitro heme polymerization assay, HRP II promoted the synthesis of hemozoin, while controls, such as the proteins bovine serum albumin and lysozyme or the homopeptides polyhistidine, polylysine, and polyasparagine, did not. FT-IR analysis of the reaction product showed the characteristic vibrations of hemozoin. The polymerization activity had a pH maximum near 4.0, which dropped off precipitously near the pKa of histidine. The heme polymerization... 

Preparations of BSA, asparaginase (aspa), alpha amylase (amy), and lysozyme (lyso) to which the indicated number of 1-thioglycopyranosides had been attached by amidi-nation. 6 Determined by the inhibition assay described in Ref. 97. c RIP = relative... 

The assay principle is straightforward the clearance of a Micrococcus hocus suspension caused by lysozyme is monitored at 450 nm and compared to a lysozyme standard of known activity. Expression of lysozyme activity in absolute terms is dependent on the substrate sensitivity. Moreover (he light scattering effect of the suspension is determined by the optical geometry of (he spectrophotometer. Therefore, absolute turbidity values can only be obtained by calibration of the apparatus with a standard of known turbidity. 

The assay of the standard and unknown lysozyme should be performed with the same substrate suspension. In the assay a suspension diluted with phosphate buffer to an absorbance of 0,800 0.050 at 450 nm is used. 

Prepare 100 mL of an enzyme solution of FJP. lysozyme HC1 in bldistilled water containing approximately 4000 FJJ. nnils/mL. Use siliconized glassware to prevent adsorption losses. Use quartz cuvettes suited for UV measurements end perform a cuvette correction. Keep the stock solution at a constant temperature of 25°Q Lysozyme in aqueous solutions is very stable, however, it is recommended to renew die solution for every assay series. 

The assay can be extended for clinical use by constructing a calibration curve of absorbance decrease per minute versus enzyme concentration with known dilutions of the lysozyme standard up to 100 jig/mL (stock concentration). 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

The objective of the experiments is to compare the affinity of wild-type antibody and wild-type immunogen (chicken lysozyme) with that of wild-type antibody and mutant antigen or with that of mutant antibody and wild-type immunogen. All the assays involve incubation of a constant concentration of one reactant with varying amounts of the complementary reactant, along with estimating the concentration of either bound or free reactant by an immunochemical or enzymatic method. In each case, the assumption is made that the measurement step does not disturb the equilibrium between antigen and antibody, and it is important that this assumption be validated experimentally. We summarize below several alternative methods. 

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. 

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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