Enzymatic Activity Of Lysozyme Research Articles

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11 700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu 2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16–20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48°C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS–PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu 2+. The displacer could also be regenerated by thermal precipitation at 48°C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).

Immobilisation de biomolécules dans des microparticules de carboxyméthylcelluloses “hydrophobiquement modifiées”

This article presents the synthesis of microparticles of carboxymethylcellulose, and of their derivatives obtained by chemical modifications with hydrophobic groups (with C 16 or C 16+C 4 amines). Their physico-chemical properties (DS, swelling, etc.) as well as their retention properties were determined to test these new supports for immobilization/separation of biological active substances such as ampicilline, ampicilline/ lysozyme mixture and other proteins. The hydrophobically modified supports present a high capacity for adsorption of lysozyme, the enzymatic activity of lysozyme being maintained.

Effect of hexanol as a cosolvent on partitioning and mass transfer rate of protein extraction using reversed micelles of CB-modified lecithin

Using the affinity-based reversed micelles composed of Cibacron Blue F3G-A (CB) modified soybean lecithin, the effect of hexanol as a cosolvent on the extraction of lysozyme and bovine serum albumin (BSA) was investigated. The water concentration in the reversed micelles increased significantly with increasing hexanol concentration. The partition coefficient of lysozyme could be increased by over 12-fold by introducing hexanol of higher than 0.5 vol%. However, the transfer of BSA was hardly affected because its high molecular weight resulted in a strong steric hindrance effect. The enzymatic activity of lysozyme was nearly 100% retained after undergoing the extraction process with the CB–lecithin micelles containing 3 vol% hexanol. The partitioning isotherms of lysozyme in the CB–lecithin micelles with and without hexanol addition were expressed by the Langmuir equation. The partitioning capacity of lysozyme was nearly increased twofold by introducing 3 vol% hexanol to the CB–lecithin micelles and reached 2.12 g/l. The cosolvent hexanol revealed insignificant effect on the mass transfer rate, and in both the systems with and without hexanol, the mass transfer rate in back extraction was 5–10 times slower than that in the forward extraction. This phenomenon was similar to that in conventionally employed ionic surfactant systems. The result suggests that in the present affinity-based reversed micelles, the interfacial resistance also played a more important role in back extraction than in forward extraction.

A lysozyme in the salivary glands of the malaria vector Anopheles darlingi.

A cDNA encoding a lysozyme expressed specifically in the salivary glands of the malaria vector mosquito, Anopheles darlingi, was isolated by differential screening an adult female salivary gland library with abdomen and salivary gland cDNAs. The primary nucleic acid sequence of the cDNA contains a deduced coding region of 429 nucleotides and 5'- and 3'-end non-transcribed regions. A signal peptide of twenty-three amino acids and a mature protein of 120 amino acids are evident in the conceptual translation product. The results of RT-PCR experiments indicated that in adult mosquitoes this gene is expressed specifically in the salivary glands. Lysozyme enzymatic activity was detected in the salivary glands and abdomens of adult mosquitoes, but the pH optimum differed for each tissue and this was interpreted to indicate the presence of more than one enzyme, each being expressed in a different tissue. The salivary gland lysozyme may be involved in protection against bacterial infection in the anterior portion of the mosquito digestive tract.

Glycation ligand binding motif in lactoferrin. Implications in diabetic infection.

Lactoferrin and lysozyme are two important, naturally occurring antibacterial proteins found in saliva, nasal secretions, milk, mucus, serum and in the lysosomes of neutrophils and macrophages. Both proteins bind specifically to glucose-modified proteins bearing advanced glycation endproducts (AGEs). Exposure to AGE-modified proteins blocks the bacterial agglutination and bacterial killing activities of lactoferrin and also inhibits the bactericidal and enzymatic activity of lysozyme. Peptide mapping by AGE ligand blot revealed two AGE-binding domains in lactoferrin, and a single AGE-binding domain in lysozyme. None of these AGE-binding domains displayed any significant homology in their primary sequences; however, a common 17-18 amino acid cysteine loop motif (CX15-16C) was identified among them, which we named an ABCD motif (AGE-Binding Cysteine-bounded Domain). Similar domains are also present in other antimicrobial proteins such as defesins. Hydrophilicity analysis indicated that each of these ABCD loops is markedly hydrophilic. Synthetic peptides, corresponding to these motifs in lactoferrin and lysozyme, exhibited AGE-binding activity. Since diabetes is associated with abnormally high levels of tissue and serum AGEs, the elevated AGEs may inhibit endogenous antibacterial proteins by binding to the conserved ABCD motif, thereby increasing susceptibility to bacterial infections in diabetic individuals. These results may provide a basis for the development of new approaches to prevent diabetic infections.

Effects of high field electric pulses on the activity of selected enzymes

A compact and low cost bench-top, pulsed electric field treatment system was designed and developed. The unit consisted of a high-voltage pulse generator (≤ 30 kV) and a treatment chamber with ≤ 148 ml capacity. Over the set-up voltage range of 4–26 kV, 30 pulses (with instant charge reversal) were applied to eight different enzyme solutions using a 0.3-cm electrode distance, a 13–87 kV/cm field, 0.5-Hz pulse frequency, 2-μs pulse width and 20 °C process temperature. For some enzymes, activities were reduced after the pulse treatments: lipase, glucose oxidase and heat-stable α-amylase exhibited a vast reduction of 70–85%; peroxidase and polyphenol oxidase showed a moderate 30–40% reduction whereas alkaline phosphatase only displayed a slight 5% reduction under the conditions employed. On the other hand, the enzyme activities of lysozyme and pepsin were increased under a certain range of voltages. Electric pulse profile (instant charge reversal) played a very important role in reducing the activities of various enzymes.

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes.

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography.

Molecular Divergence of Lysozymes and α-Lactalbumin

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for α-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. α-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in α-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.

Enhanced Reactivity of Lysozyme with Formaldehyde during Cold Denaturation

The reactivity of lysozyme with formaldehyde at low temperatures (0 to -20 o C) was compared with lysozyme reactivity at 37 o C. Lysozyme reaction with formaldehyde, in the presence and absence of sodium cyanoborohydride, was also compared. The progress of reactions was evaluated by measurement of protein primary amino groups, lysozyme enzymatic activity, and occurrence of cross-linkage as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained indicated that the chemical modification of lysozyme by formaldehyde was significantly enhanced at cold freezing temperatures (-6 and -10 o C) when compared with 37 o C

A rapid microtiter plate method for the detection of lysozyme release from human neutrophils

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng ml . Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC 50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC 50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t- Boc- l-methionyl- l-leucyl- l-phenylalanine ( N-t-Boc) , LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC 50 of 2 μM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC 50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC 50 of 2 μM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively. The adaptation of this assay, as demonstrated here, to a microplate format provides a simple, reproducible, and high throughput method that is ideally suited for largescale drug screening.

Characterization of complex formation between lipopolysaccharide and lysozyme

The binding of lysozyme (LZM) to bacterial lipopolysaccharide (LPS) inhibited the biological activities of LPS as well as the enzymic activity of LZM. The mode of binding has been characterized by using dansylated LZM and enzyme inhibition. The binding of LPS to LZM significantly increased the fluorescence intensity (Fl-intensity) of the danyl group and was found to be time-dependent; the complex was produced gradually and became stabilized within 20 min at 37°, 10 min at 50°, and 1 min at 70°. The maximum level of binding was also dependent on the reaction temperature, and more complex was formed at higher temperatures. Complexation was strongly dependent on the salt concentration and was not observed at >0.5 m NaCl. From collected evidence of the Fl-intensities of various dansyl derivatives and amphiphiles, it is concluded that LZM interacts with LPS by multiple binding-modes, the first being strongly related to the enzyme inhibition, the second being close to the Fl-intensity, and the third being dependent on the inhibition of immunopharmacological activities. For the amphiphiles used in this study, sdium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-propanesulfonate (CHAPSO), decansulfonic acid, and cardiolipin have binding modes similar to that of LPS.

Lipopolysaccharide interaction with lysozyme: Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity.

Experiments have been carried out to characterize the binding of lysozyme (LZM) to bacteriol lipopolysaccharide (LPS). The formation of LPS·LZM complexes can be readily demonstrated using either physical-chemical separation techniques or a radiolabeled photoaffinity LPS probe. The binding affinity of LZM for LPS has been estimated to be ∼ 108 liters/mol. Binding of LPS results in loss of LZM enzymatic activity by a noncompetitive inhibition, as assessed by either particulate or soluble substrates. This interaction of LPS with LZM is dictated primarily by hydrophobic interactions and appears to be a general property of both constituents. Binding can be demonstrated with LZM of both human and avian sources, as well as with LPS isolated from a variety of Gram-negative organisms. The addition of LPS to biologically relevant fluids containing LZM results in dose-dependent inhibition of LZM enzymatic activity suggesting that such interactions may have relevance in Gram-negative infections. Finally LZM has been shown to reduce the endotoxic activity of LPS as assessed by gelation of Limulus amoebocyte lysates.

Studies on human dental plaque. 1. Physical and chemical characteristics and enzyme activities of pooled plaque extracts.

Human supragingival dental plaque was collected from patients with various degrees of caries and periodontal disease. Plaque extracts, prepared in five different solutions (four varied from pH 1.8 to 12.7; one contained urea), were analyzed by polyacrylamide gel electrophoresis, and tested for amylase and lysozyme enzyme activity. Because no qualitative or quantitative advantages of using the extremes of pH or urea were observed, all subsequent extracts were prepared in phosphate buffered saline at pH 7.3. Concentrated extracts were fractionated by gel filtration and characterized by polyacrylamide gel electrophoresis, peptide mapping, molecular weight estimation, determination of enzymatic activities and amino acid and carbohydrate analyses. Regions of similarity among the gels were revealed by comparing the electrophoretic patterns of pooled plaque extract, normal serum and whole saliva. The elution pattern of pooled plaque extract from a standardized Sephadex G-200 column indicated the presence of both high and low molecular weight proteins that might have correlated with the components of normal serum and saliva. A predominant and dialyzable third fraction had no correlate in either serum or saliva. The small peptides in this fraction were subjected to amino acid, carbohydrate and peptide map analyses. The most abundant amino acids were alanine, glutamic acid, glycine, valine, leucine, lysine and serine. These small components contained no neutral or amino sugars. Pooled plaque extract and the small peptides exhibited similar peptide maps.

STUDIES ON LYSOZYMES VI

The enzymic activity of lysozyme has been colorimetrically determined by 3, 4- dinitrophenyl-tetra-N-acetyl-β-chitotetraoside. The hydrolytic reaction of the sub-strate by human milk lysozyme is most effective in the buffer, pH 6, with Na+ concentration of 0.1 M. The hydrolytic activities of three human lysozymes and hen egg white lysozyme have been measured by the colorimetric assay.

Identification of essential residues in lysozyme: a pulse radiolysis method.

THE technique of pulse radiolysis has recently been applied to the study of the radiation inactivation of the enzyme lysozyme1. While pursuing this technique we devised the following method of identifying some of the amino-acid residues associated with the enzymatic activity of lysozyme. The results show that specific free-radical attack at a tryptophan residue destroys the enzymatic activity and is consistent with crystallographic evidence2 for the crucial role of tryptophan at the active site.

Studies on the enzymic activity of lysozyme, 3. The binding of saccharides.

Studies on the enzymic activity of lysozyme, 3. The binding of saccharides.

STUDIES ON THE ENZYMIC ACTIVITY OF LYSOZYME, II. THE HYDROLYSIS AND TRANSFER REACTIONS OF N -ACETYLGLUCOSAMINE OLIGOSACCHARIDES

STUDIES ON THE ENZYMIC ACTIVITY OF LYSOZYME, II. THE HYDROLYSIS AND TRANSFER REACTIONS OF <i>N</i> -ACETYLGLUCOSAMINE OLIGOSACCHARIDES

Influence of H 2 S on the Radiosensitivity of Enzymes and Microorganisms

The purpose of the work herein described was to study the effects of H2S on the radiosensitivity of enzymes and microorganisms exposed in aqueous solution. Two different effects were studied-the inactivation of the enzymatic activity of lysozyme, and the inactivation of the colony-forming ability of bacteria.

The role of tryptophan residues in the enzymic activity of lysozyme

The role of tryptophan residues in the enzymic activity of lysozyme

Reversible inhibition of enzymes by interaction with synthetic polysaccharide macroanions

Charged polyglucose derivatives, such as polyglucose sulfates, with different molecular weights, branching, and degree of substitution precipitated basic proteins, such as lysozyme, ribonuclease, protamine, thymus peptide, polymyxin B, an ACTH preparation, cytochrome c, and serum albumin from aqueous solution at low pH and salt concentration. Enzymic activity of lysozyme, ribonuclease (both on ribonucleic acid and on a cyclic phosphate), and hyaluronidase was inhibited in solution. The inhibition required macromolecular size and polyanionic structure and depended on the type and amount of charged groups on the synthetic polysaccharide derivative. Highly substituted sulfates were the most effective inhibitors, while sulfates with similar molecular size but lower charge density or derivatives with weaker dissociating anionic groups (carboxyls) had less inhibitory activity. The inhibition appears to be due to electrostatic interaction forces between the oppositely charged macromolecules and can be reversed by a polymeric cation stronger than the substrate.