Lysozyme chromatography

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

Yang, Q. Lundahl, P., Binding of lysozyme on the surface of entrapped phosphati-dylserine-phosphatidylcholine vesicles and an example of high-performance lipid vesicle surface chromatography, J. Chromatogr. 512, 377-386 (1990). 

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

M.Y. Arica, M. Yilmaz, E. Yalcin and G. Bayramoglu, Affinity membrane chromatography relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification. J. Chromatogr.B, 805 (2004) 315-323. 

Dismer F. Petzold M. Hubbuch J. Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents. Journal of Chromatography A, 2008, 1194, 11-21. 

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

Chromatographic Analysis. The samples of native and ozonized lysozyme (lysozyme treated with ozone just to the point of complete inactivation) were analyzed by column chromatography. The column (0.8 X 56 cm.) containing DEAE-Sephadex A-50 (Cl form) resin, was equilibrated with 0.1 M Tris Cl buffer, pH 8.3, and loaded with about 2-U mg of protein. Aliquots eluted with 0.1 M Tris-Cl pH 8.3 were collected and absorbance at 278 nm was measured. The native lysoz3mie eluted earlier than the ozonized products. This difference may be assoicated with both aggregation of protein and ionic behavior of the residues. 

In order to analyse the properties of the inactivated lysozyme, the enzyme was subjected to further analysis by electrophoresis and chromatography. Both analyses of the samples at various pHs, inactivated to the extent over 95%, indicated that the product is composed of one peak. The ozonized lysoz3mie moved slower than the native lysozyme on DEAE-Sephadex and the products at different pHs were readily distinguished from each other (Fig, 3), However, a diffuse band was observed for ozonized lysozyme as distinct from a sharp band for native lysozyme in polyacrylamide gel (17), The presence of only one band suggests that the ozonolysis does not cause the cleavage of peptide bonds and the remaining activity is not due to the presence of a small amount of unmodified lysozyme. 

Figure 3. Chromatography of native and ozonized lysozyme on DEAE Sephadex. Samples of lysozyme at the three different pHs were inactivated 95-100% by ozone. The samples were applied to a column of DEAE-Sephadex A-50 (0.8 X 56 cm) and eluted with O.OIM Tris-HCl pH 8.3 and a gradient of O.OIM NaCl. Fractions of 2 ml were collected. The results of four columns are plotted.

An example of the application of RARE for rapid image acquisition is shown in Fig. 19, in which a single frame is shown from a series of 2-D images acquired of an oscillatory chemical reaction occurring within a fixed bed. Relaxation contrast is used to discriminate between the reaction products Mn " and Mn (49). In this example, MR offered the opportunity to map the detailed structure of the fixed bed and the product distribution within it. This pulse sequence has also recently been applied to obtain quantitative images of the evolution of a lysozyme urea separation within a chromatography column (50). 

Selective adsorption on glass Ammonium sulfate fractionation Ammonium sulfate fractionation Ethanol precipitation Sonic disintegration Selective adsorption on cellulose phosphate CM-cellulose chromatography Acetone precipitation Ammonium sulfate fractionation Ethanol precipitation Selective adsorption on cellulose phosphate Ammonium sulfate fractionation Lysozyme and trypsin digestion Ammonium sulfate fractionation DEAE-cellulose chromatography Ammonium sulfate fractionation... 

Figure 2.9 Hydrophobic-interaction chromatography of proteins. (A) Ammonium sulfate gradient from 2.16 to 0 M (B) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 10 mM, respectively (C) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 20 mM, respectively (D) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 40 mM, respectively. Chromatography conditions column, silica-bound polyether, 10 cm x 4.6 mm I.D. temperature, 25°C flow rate, 1 ml/min gradient, linear for 30 min background buffer, 50 mM phosphate, pH 6.5. Peaks a, cytochrome c b, ribonuclease A c, /3-lactoglobulin A d, lysozyme e, ovalbumin f, a-chymotrypsinogen A g, fetuin. (Reprinted from Ref. 45 with permission.)...

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