Alkali lysozyme

In concentrated NaOH, chitin becomes alkali chitin which reacts with 2-chloroethanol to yield 0-(2-hydroxyethyl) chitin, known as glycol chitin this compoimd was probably the first derivative to find practical use (as the recommended substrate for lysozyme). Alkali chitin with sodium monochloroacetate yields the widely used water-soluble 0-carboxymethyl chitin sodium salt [118]. The latter is also particularly susceptible to lysozyme, and its oUgomers are degraded by N-acetylglucosaminidase, thus it is convenient for medical appHcations, including bone regeneration. 

The chemical barriers are also important but are sometimes neglected in discussions of defence. To some extent they are specific for each tissue or organ and examples are as follows. Enzymes, such as lysozyme, which digests part of the bacterial coat, are present in many secretions (e.g. tears, milk) acid in the stomach, in the vagina, on the surface of the skin alkali in the small intestine mucus in the intestine, respiratory tract and vagina. 

This volume has been written for those chemists and biochemists who may never themselves do X-ray diffraction analyses of crystals, but who need to be able to understand the results of such studies on structures of immediate interest to them. The fields of structural biology and chemistry have blossomed in the years since X-ray diffraction was discovered in 1912. For example, the three-dimensional structures of benzene, graphite, the alkali halides, the boron hydrides, the rare gas halides, penicillin, vitamin Bjj, hemoglobin, lysozyme, transfer RNA, and the common-cold rhinovirus have been determined and, in each case, the results have greatly increased our understanding of fundamental chemistry and biochemistry. 

The effects of alkali up to 0.1 N on the absorption spectrum of lysozyme are fully reversible, according to Fromageot and Schnek (1950) it is of interest to note that this enzyme is a protein which is remarkably resistant to denaturation (Fraenkel-Conrat, 1950). Insulin behaves similarly, and it may be surmised that in both these proteins the tyrosine hydroxyl groups are not bound. The pK value of 10.8 found by Fromageot and Schnek (1950) for lysozyme supports this view. The pK data for insulin are more complicated and have been discussed at length by Crammer and Neuberger (1943). 

Time effects have also been observed for bovine plasma albumin, horse serum albumin and rabbit y-globulin (Beaven and Holiday, 1950). The behavior of bovine plasma albumin was most striking the intensity of the absorption in 0.1 N alkali increased steadily over a period of ca. 3 hours at room temperature the light-scattering properties of the solution, as shown by its apparent absorption on the long-wave side of the absorption band proper, also increased. Of the few proteins which were examined, only lysozyme showed no time effect. With trypsin the change in absorption was small and complete in a few minutes at pH 13, but the solution then became visibly turbid. 

Hayashi and Kameda have reported 40% to 70% decreases in pepsin-catalyzed hydrolysis of lysozyme, soybean protein, casein and rlbonuclease A due to alkali-treatment under slightly milder conditions than ours (16, 29). Friedman, Zahnley and Masters reported an 80% decrease in digestibility of sodium hydroxide-treated casein measured as hydrolysis by trypsin (17). However, trypsin is specific for lysyl residues and lysine levels decreased to about half control values during the alkali-treatment, with a concomitant Increase in LAL formation. The somewhat lower digestibilities reported by these laboratories compared to our observations may be due to LAL formation in the proteins other than zein. 

The A. coerulea material was poorly crystalline, showing a broad peak at 20° 20, but the spectrum for the alkali-treated material showed a broad peak at 10.72° and two peaks at 18.72° and 19.98° 20, with close similarity to the spectrum of authentic chitosan. Optical microscopy showed that the alkali-treated products, stained with Saphranine or with other stains, preserved the morphology of the fungus, with flattened and empty structures [30] (Fig. 1). This work introduced the concept that an extended surface area of the carbohydrate polymer leads to enhanced performance, as amply confirmed by most recent works dealing with chitin and chitosan nanofibrils. In fact, the partially re-acetylated chitosan (degree of acetylation 0.23) is promptly depolymerized by lysozyme, papain, and lipase thanks to the ideal degree of acetylation for maximum enzymatic activity. Remarkably, the re-acetylated... 

Strong acids cleave C. into D- glucosamine (chitosa-mine) and acetic acid. On decomposition with alkalis, acetates and the weakly basic deacetylated, partially depolymerized, and crystallizable chitosan are formed the latter can form gels and films. The chiti-nases (EC 3.2.1.14) found in snail stomachs, some mold fungi, and bacteria can - like some lysozymes -dissolve C. The thus formed chitobiose, the disaccharide of P-1,4-linked NAG, is then cleaved to monomers by chitobiase (EC 3.2.1.30). Tlie formation of C. is catalyzed by the enzyme chitin synthase (EC 2.4.1.16). The activity of, e.g., the insecticide diflubenzurone is based on inhibition of this enzyme. 

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points.The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only L-tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the L-tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried L-tyrosyl residue on alkali denaturation. The disulphide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of L-tyrosine-23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 C from the c.d. titration data. Comparison of the c.d. 

The cited evidence for the B-elimination mechanism leading to dehydroalanine formation merits further comment. Nashef et al. (41) report that alkali-treatment of lysozyme ribonuclease and several other proteins resulted in loss of cystine and lysine residues and the appearance of new amino acids lysinoalanine, lanthionine, and B-aminoalanine. Alkali-treatment of the proteins induced an increase in absorbance at 241 nm, presumably from the formation of dehydroalanine residues. The dehydroalanine side chain can participate in nucleophilic addition reactions with the e-NH2 group of lysine to form lysinoalanine, with the SH groups of cysteine to form lanthionine, and with ammonia to form B-aminoalanine. 

Chitin-coated cellulose (produced by treating cellulose with alkali-chitin) has been used in the purification of lysozyme and a derivative of lysozyme by affinity chromatography. DNA derivatives of cellulose have been used in the chromatography of phosphorylated, nucleolar nonhistone and in the purification of a glucocorticoid receptor from rat liver. 

Chondroitin 4-sulphate and cartilagenous proteoglycans form ionic complexes with lysozyme. The complexes are solubilized by either salts or alkali, although a higher salt concentration is needed to solubilize the proteoglycan complexes. The distribution of charges on the complexes and the implications of the formation of ionic complexes in tissues were discussed. 

Linear chain polymers exist as a polysaccharide protein in the organism. Specific rotation [a]D = -15° (in concentrated hydrochloric acid). White powder. When completely hydrolyzed in acid, it obtains a D-glucosamine acetic acid. When in alkali, decomposes to chitosan and acetic acid. Disassociates by enzyme chitinase of snails and worms or lysozyme and creates an N-acetyl-D-glucosamine or its oligosaccharides. 

---