Carbon lysozyme

Carbon atom, 4. See also Atomic orbitals Carbon dioxide hydration, 197-199. See also Carbonic anhydrase Carbonic anhydrase, 197-199,200 Carbonium ion transition state, 154, 159 Carboxypeptidase A, 204-205 Catalysis, general acid, 153,164,169 in carboxypeptidase A, 204-205 free energy surfaces for, 160, 161 in lysozyme, 154... 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

Adsorption of lysozyme on these mesoporous carbon materials was studied at a solution pH of 11 [152]. All the adsorption isotherms were of a Langmuir type (see Figure 4.11A), resulting in monolayer adsorption capacities of 3.8, 9.8, 15.9, and 22.9 pmol g 1 for CMK-1, CMK-3, CMK-3-130, and CMK-3-150, respectively. These values are compared with the structural parameters of the CMK materials (Figure 4.11B). There is no clear relation between the adsorption capacity and the surface area (Figure 4.11B(a)). In sharp contrast, positive correlation between the adsorption... 

Fig. 4.11 (A) Adsorption isotherms of lysozyme meters of the CMK materials (a) specific surface on CMK-type mesoporous carbon materials at pH area (b) specific pore volume (c) pore diameter. 11 (a) CMK-1 (b) CMK-3 (c) CMK-3-130 (d) Reprinted with permission from [152], A. Vinu CMK-3-150. (B) Dependence of the adsorption etal.,J. Phys. Chem. B 2005, 109, 6436. 2005, capacities of lysozyme on the structural para- American Chemical Society.

These excellent textural characteristics would be very useful for the adsorption of biomolecules. Figure 4.17 compares the adsorption behavior of lysozyme on CMK-3 (plot (a)), CMK-3-150 (plot (b)), and the carbon nanocage (plot (c)) at pH 11 [154],... 

Fig. 4.17 Adsorption isotherms of lysozyme on various carbon materials (a) CMK-3 (b) CMK-3-150 (c) carbon nanocage.

We found that solutions of hen egg white lysozyme, bovine ribonuclease A (RNase A), or a 1 2 mol ratio of bovine carbonic anhydrase lysozyme formed opaque gels within 2 min when mixed with an equal volume of 20% NBF.25,26 Multi-protein tissue surrogates comprised of 50% w/v lysozyme and up to four additional proteins have also been formed (Fowler et al., unpublished results). After overnight fixation, the surrogates were firm and sliced easily with a razor blade for sampling. To determine the optimal... 

Notes-. Tissue surrogate samples (1.5 mg) histologically processed to paraffin embedding were rehydrated and resuspended in recovery buffer (20 mM Tris-HCl+2% SDS) at the indicated pH. Total protein in the supernatants was assessed colorimetrically after heating at 100°C for 20 min, followed by 60°C for 2h. The two-protein tissue surrogates were composed of carbonic anhy-drase lysozyme (2 1 mol/mol). The % recovery values are the mean, the standard deviation. For more detail, see Reference 25. 

Figure 14.4 Gel image of proteins extracted from a mixed carbonic anhydrase lysozyme tissue surrogate. Lane M, molecular weight marker lane 1, a 1 2 mol ratio mixture of native, non-formalin-treated carbonic anhydrase and lysozyme lane 2, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 4.0, with 2% SDS lane 3, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 6.0, with 2% SDS. Protein bands corresponding to lysozyme monomer (a), carbonic anhydrase monomer (b), and the putative lysozyme-carbonic anhydrase heterodimer (c) are indicated. For more detail, see Reference 25.

Fig. 3 SDS-PAGE Photograph Separation (Lane Mr and A) and activity staining (Lane B and C) of the crude filtrate of Funalia trogii. Lane Mr standard molecular weight markers ([3-galactosi-dase, 118.0 kDa bovine serum albumin, 79.0 kDa ovalbumin, 47.0 kDa carbonic anhydrase, 33.0 kDa P-lactoglobulin, 25.0 kDa and lysozyme, 19.5 kDa). Relative mobilities of the standard markers vs. common logarithms of their molecular masses were plotted.With the linear regression output, the molecular masses of the proteins in the crude filtrate were estimated (taken from [18])...

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...

If aspartic acid-52 acts as a nucleophile in lysozyme reactions a glycosyl enzyme intermediate will be formed [60]. There is no evidence, kinetic or otherwise, for substituted enzyme intermediates, but rapid breakdown might preclude attainment of detectable concentrations. Formation of a substituted enzyme could explain the observed retention of configuration at the anomeric carbon in transglycosidation reactions, provided backside attack in a subsequent reaction is chemically reasonable. It has therefore been important to attempt to understand the chemistry of acylal hydrolysis so as to assess the properties that would be expected of an acylal intermediate in reactions catalysed by the enzyme. 

Fig. 2.1. Semilogarithmic plot of molecular weight (Mr) of marker proteins vs relative mobility (Rf) of marker proteins in gels of different acrylamide concentrations %T. Proteins 1 aprotinin (6.5 kD) 2 lysozyme (14.5 kD) 3 soybean trypsin inhibitor (21.5 kD) 4 carbonic acid anhydrase (31 kD) 5 hen ovalbumin (45 kD) 6 bovine serum albumin (66 kD) 7 phosphorylase b (97.4 kD) 8 8-galactosidase (116 kD) 9 myosin (205 kD)...

In addition to red-shifted spectra and low rotational amplitude, Ehrlich ascites plasma membranes exhibit Cotton effects in the region of aromatic chromophores, 250-300 m/ (89). Similar behavior has been observed in other proteins, such as carbonic anhydrase (34) and lysozyme (58), and disappear upon denaturation. Ehrlich ascites plasma mem-... 

Lysozyme and /3-galactosidase, which are both glycosidases, catalyze very similar reactions. Both enzymes are found to catalyze the alcoholysis of their polysaccharide substrates with retention of configuration at the C-l carbon (equation 8.25).14-17 This is consistent with the evidence presented in Chapter 7, section C3, that there is at least one (but probably only one) intermediate on the reaction pathway. However, kinetic isotope data are consistent with the interpretation that the intermediate in the reaction of /3-galactosidase is covalent and that there are two successive SN2 displacements, whereas the intermediate with lysozyme is a bound carbonium ion formed in an SN1 reaction (Chapter 16). The carbonium ion, unlike an analogous one in solution, reacts stereospecifically on the enzyme. Thus, the stereochemical evidence by itself has given no indication of the nature of the intermediate. 

Using 20 mm tubes the sensitivity of natural abundance PFT 13C NMR can be increased drastically, as was demonstrated first for hen egg-white lysozyme. For example, the 13C NMR spectrum recorded under these conditions shows 22 signals for 28 non-protonated aromatic carbons, however, with a broad background arising from 59 proto-nated aromatic carbons [916]. 

To test this idea, a Bio-Rad Protean II electrophoresis cell and Bio-Rad Model 3000xi computer-controlled power supply were used to carry out the electrophoretic separation and recovery of pre-stained and unstained protein calibration standards (lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B) obtained from Bio-Rad Laboratories. Standard SDS-gel electrophoresis techniques were used [167]. 

Figure 25-29 shows an unusually well-resolved 13C nmr spectrum of the enzyme lysozyme (Table 25-3 and Figure 25-15) taken with proton decoupling. The closely spaced peaks on the left side of the spectrum are of the carbonyl groups. The peaks in the center are of unsaturated and aromatic carbons, while those on the right are of the aliphatic amino acid carbons. The five sharp resonances marked at about 110 ppm with arise from tryptophan carbons marked with in 22 ... 

Figure 25-29 Carbon-13 nmr spectrum at 45.3 MHz of lysozyme, 0.015M in water solution, taken with proton decoupling (Section 9-10L)...

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