Description
Introduction: Lysozymes, also known as muramidase or N-acetylmuramide glycanhydrolase, are a family of enzymes which damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. It is abundant in a number of secretions, such as tears, saliva, and mucus. Lysozyme is also present in cytoplasmic granules of the polymorphonuclear neutrophils (PMN). Large amounts of lysozyme can be found in egg whites. C-type lysozymes are closely related to alpha-lactalbumin in sequence and structure making them part of the same family. In humans, the lysozyme enzyme is encoded by the LYZ gene. The enzyme functions by attacking peptidoglycans and hydrolyzing the glycosidic bond that connects N-acetylmuramic acid with the fourth carbon atom of N-acetylglucosamine. It does this by binding to the peptidoglycan molecule in the binding site within the prominent cleft between its two domains. This causes the substrate molecule to adopt a strained conformation similar to that of the transition state. According to Phillips-Mechanism the lysozyme binds to a hexasaccharide. The lysozyme then distorts the 4th sugar in hexasaccharide (the D ring) into a half-chair conformation. In this stressed state the glycosidic bond is easily broken. 3 The amino acid side chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whilst Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The glycosyl enzyme intermediate then reacts with a water molecule, to give the product of hydrolysis and leaving the enzyme unchanged.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with a goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated LZM and antibody preparation specific for LZM and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of LZM in the samples is then determined by comparing the O.D. of the samples to the standard curve