Assay of lysozyme

Other methods have considerably less detectability. The photometric rate-of-lysis assays (Shugar, 1952 Selsted and Martinez, 1978 Carroll and Martinez, 1979) detect 20 ng of lysozyme in a matter of minutes, but suffers from a limited range of linearity. The so-called lysoplate assay (Osserman and Lawlor, 1966) is about 10 times 

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Inaba, T., Ohguchi, T., Iga, T., and Hasegawa, E. 1984. Synthesis of 4-methylcoumarine-7-yloxy tetra-A -acetyl-P-chitotetraoside, a novel synthetic substrate for the fluorometric assay of lysozyme. Chem. Pharm. Bull. 32, 1597-1603. 

Fig. 43. Assay of lysozyme activity following release from a polymer prepared from 1,2,6-hexa-netriol and triethyl orthoacetate at pH. 7.4 and room temperature. Activity assayed by noting change in rate of lysis of Micrococcus lysodeikticus. Lysozyme loading 10 wt% [50]. Reprinted with permission...

Assays of lysozyme by a diffusion technique, which employs an agarose gel impregnated with substrate organisms (lysoplates), differed greatly from those obtained by spectrophotometric and immunological techniques. Different agaroses had unpredictable effects on the determination of lysozyme in normal... 

Methylumbelliferyl tri-JV-acetyl-j8-chitotrioside is a convenient substrate for the assay of lysozyme, since the 4-methylumbelliferone formed can be measured by the increased fluorescence at 442 nm. The binding constant and rate of hydrolysis of this trioside were higher than those of 4-nitrophenyl tri-iV-acetyl-/5-chitotrioside. 4-Methylumbelliferyl glycosides of 2-acetamido-2-deoxy-j8-D-glucopyranose and di-A acetyl-jS-chitobiose were also investigated as substrates. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

Figure 1. Inactivation of lysozyme by ozone. Volumes of 4.8 ml of O.IM buffer containing 2.814 mg of lysozyme were exposed to a gas stream of ozone in oxygen (1.8 nmole/min ozone). Aliquots were removed for enzyme assay as described in Materials and Methods.

The assay principle is straightforward the clearance of a Micrococcus hocus suspension caused by lysozyme is monitored at 450 nm and compared to a lysozyme standard of known activity. Expression of lysozyme activity in absolute terms is dependent on the substrate sensitivity. Moreover (he light scattering effect of the suspension is determined by the optical geometry of (he spectrophotometer. Therefore, absolute turbidity values can only be obtained by calibration of the apparatus with a standard of known turbidity. 

The assay of the standard and unknown lysozyme should be performed with the same substrate suspension. In the assay a suspension diluted with phosphate buffer to an absorbance of 0,800 0.050 at 450 nm is used. 

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

Two different sequential saturation assays are modified from the ELISA25 to determine association constants of antibody with lysozymes. In both assays, a constant antibody concentration is incubated with varying concentrations of lysozyme until equilibrium is reached. The time necessary must be determined empirically for each antibody for the antibody HyHEL-10, this was determined to be at least 16 hr,26 and all incubations were performed for 16-24 hr. At the end of the incubation, an aliquot of the mixture is treated with an excess of reagents to sample free (unbound) antibody combining sites by incubating with labeled or solid-phase coupled... 

The culture is harvested by centrifugation at 5000 g and 4° at peak production. Lysozyme activity, which is monitored by the standard turbidity assay of culture supernatant (described in a later section), typically reaches its peak after 5 to 7 days of growth in rich medium. For inactive mutants, the peak of lysozyme secretion is estimated as 12 to 24 hr after the yeast cell density (monitored by A6S0) reaches its peak. 

Twenty-five 1-ml fractions of the eluent are collected and assayed for lysozyme activity. The fractions containing lysozyme activity (typically 6) are pooled. 

The study by Determan et al. [224] focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of TT, OVA, and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride [sebacic acid and l,6-bis(p-carboxyphenoxy)hexane] monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. SDS-PAGE, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. ELISA was used to measure changes in the antigenicity of TT and OVA and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. TT toxoid was found to be the most stable in the presence of anhydride monomers, while OVA was most stable in the... 

Delfini, C. (2004). Resistance screening assay of wine lactic add bacteria on lysozyme efficace of lysozyme in unclarified grape musts. J. Agric. Food Chem., 52, 1861-1866. 

Numerous reviews have documented immune modulation in response to metal, pesticide and organic contaminants in fish4,19,37,135. Assays traditionally used to assess perturbations of immune function in fish fall into three broad categories pathogen challenge models, assays that monitor immune suppression/activation or immuno-pathology. Immune suppression/activation is usually measured with assays of phagocytosis, respiratory burst, cell proliferation, as well as quantification of soluble factors such as lysozyme, serum antibody, CRP or complement. 

Stability studies of lysozyme immobilized in a polymer where R is methyl are shown in Fig. 43 [50]. The activity of the released lysozyme was assayed by noting rate of lysis of Micrococcus lysodeikticus [57], The control was a solution made up at the start of the experiment and stored at room temperature. This solution was sampled at each time point of the release experiment. 

Brodbeck and Ebner found that the soluble lactose synthetase from milk can be separated into two protein components, A and B, which individually do not exhibit any catalytic activity however, their recombination restores full lactose synthetase activity. The B fraction has been crystallized from bovine skim milk and bovine mammary tissue, and was identified as a-lactalbumin. It was thus found that a-lactalbumin can be substituted for the B protein of lactose synthetase. Lactose synthetases from the milk of sheep, goats, pigs, and humans were also resolved into A and B proteins, and the fractions from these species were shown to be qualitatively interchangeable in the rate assay of lactose synthesis. Determination of the amino acid sequence of a-lactalbumin (B fraction) has shown a distinct homology in the sequence of amino acids of a-lactalbumin and hen s egg-white lysozyme, suggesting that lysozyme and a-lactalbumin have evolved from a common ancestral gene. 

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