Lysozyme, solution preparation

Cordova et al. explored the CE behavior of protein charge ladders obtained by acetylation of lysozyme and carbonic anhydrase II using noncovalent polycationic coated capillaries. Two of them, polyethylenimine and Polybrene, were very effective in preventing the adsorption of positively charged proteins. Conditions used were fused-silica capillary (50 p.m i.d. x 38 cm) coated with the polymer by flushing the capillary with a 7.5% (w/v) polymer solution, prepared in 25 mM Tris-192 mM Gly buffer (pH 8.3), for 15 min. The running buffer was 25 mM Tris-192 mM Gly buffer (pH 8.3) in the absence of polymer. Separations were obtained within 5 min. 

FIGURE 10.12 Theoretical and experimental nucleation rate of lysozyme crystals as a function of the Wenzel roughness coefficient (lysozyme dissolved in 0.05 M NaAc buffer at pH 4.5 a precipitant solution prepared by dissolving NaCl in NaAc buffer. Both solutions filtered and mixed 1 1 to give a final NaCl concentration of 2 wt% a final protein concentration of 40 mg/ml). (Adapted with permission from Cutcio et al., J. Phys. Ghent. B, 114,13650-13655. Copyright 2010 American Chemical Society.)... 

The hen egg white lysozyme protein sample with molecular weight of 14,300 and nonprosthetic groups used in this work was obtained from Wako Chemical Co.. The aqueous lysozyme solutions with a wide range of protein concentrations from 2 to 200 wt/wt% were prepared in an NMR tube with a diameter of 5 mm. labeled water (20%) was obtained from Cambridge Isotope Laboratories. 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Figure 14.3 SDS-PAGE of recovery of lysozyme in the presence of BME. Lane M, molecular weight marker lane 1, FFPE lysozyme tissue surrogate lane 2, a 75 mg/mL solution of lysozyme heat coagulated for 10 min at 100°C in 10 mM sodium phosphate buffer, pH 7.4. Both preparations were resuspended in 20mM Tris-HCl, pH 4, with 2% SDS and 0.5 M BME, and heated at 100°C for 20min followed by a cycle of heating at 60°C for 2h. For more detail, see Reference 25.

Characterization of Enzyme-Substrate Complex by use of CM-Chitin (Carboxymethyl Chitin). CM-chitinwas prepared by carboxymethylation of chitin according to the method of Imoto, Hayashi and Funatsu (13). The ozonized lysozyme (1.3 mg) solutions at different pHs were neutralized with NaOH or HCl to pH 8.0 and the poured into the column (1.5 X 4 cm.) containing white cotton-like Cm-chitin ( 65 mg.), which was equilibrated with 0.1 M Tris-Cl buffer pH 8.0. Aliquots were eluted first with 0.1 M Tris-Cl pH 8.0 and then with 0.2 M HAc. The absorbance of the fractions was measured spectrophotometrically at 280 nm. 

Figure 3.3 Schematic map of crystallization kinetics as a function of lysozyme and NaCI concentration obtained from a matrix of dishes. Inserts show photographs of dishes obtained 1 month after preparation of solutions. From G. Feher and X. Kam, in Methods in Enzymology 114, H. W. Wyckoff, C. H. W. Hirs, and S. N. Timasheff, eds., Academic Press, Orlando, Florida, 1985, p. 90. Photo and caption reprinted with permission.

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. 

Disposable cuvettes are used only once to eliminate the possibility of lysozyme carryover. All microcentrifuge tubes and pipette tips are autoclaved, and buffers and BSA solutions are not used beyond 7 days. The M. lysodeikticus suspension is prepared 18-24 hr before use and set at 37° with shaking, to hydrate fully. It was found that substrate prepared just before use settled much faster and interfered with the signal at the low concentrations of enzyme used. 

Standard Preparation Use a commercial reference standard lysozyme of a specified strength from an animal or microbial source in accordance with the origin of the preparation being measured. Measure 50 mg of the reference standard lysozyme into a 50-mL volumetric flask, and dissolve, with stirring, in approximately 25-mL of Sodium Phosphate Buffer Solution. Dilute to volume with Sodium Phosphate Buffer Solution, and mix thoroughly. If desired, freeze aliquots of this Standard Preparation for subsequent assays. Quantitatively transfer 3 mL of the Standard Preparation to a 100-mL volumetric flask, and dilute to volume with Sodium Phosphate Buffer Solution. 

Protein molecular weight standards—Suggested standards are phosphorylase (97,400 Da), bovine serum albumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da) and lysozyme (14,400 Da) at 1 mg/ml each in a single mixture in 0.01 M Tris chloride buffer, pH 7.0 (prepared by dilution of the stock 1 M buffer above). Approximately 2 ml of this solution will be required. 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

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