Enzyme-linked proteins covalently

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

The enzymes are protein molecules having globular structure, as a rule. The molecular masses of the different enzymes have values between ten thousands and hundred thousands. The enzyme s active site, which, as a rule, consists of a nonproteinic organic compound containing metal ions of variable valency (iron, copper, molybdenum, etc.) is linked to the protein globule by covalent or hydrogen bonds. The catalytic action of the enzymes is due to electron transfer from these ions to the substrate. The protein part of the enzyme secures a suitable disposition of the substrate relative to the active site and is responsible for the high selectivity of catalytic action. 

Disulfide bond The side chain of cysteine contains a sulfhydryl group (-SH), which is an important component of the active site of many enzymes. In proteins, the -SH groups of two cysteines can become oxidized to form a dimer, cystine, which contains a covalent cross-link called a disulfide bond (-S-S-). (See p. 19 for a further discussion of disulfide bond formation.)... 

Enzyme-Linked Immunosorbent Assays (ELISAs) These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an ezyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested. 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

Entrapment of enzymes and cells has played an important role in developing bioprocesses. Applications of entrapment technology to biosensors and bioanalysis have mainly been focused on udlizadon of cells and, to a smaller extent, on enzymes (24). Combining covalent coupling and entrapment cross-links enzymes and inert protein to form a protein membrane that covers the sensitive part of the electrode dp in bioanalytical applications (25). Entrapping enzyme aggregates is another variadon of this methodology (26). 

The site-specific modification of enzymes with a single electron-relay group located near to the redox cofactor and providing efficient electrical contact with the conductive support has been achieved by the reconstitution of enzymes with cofactors covalently linked to redox groups. Affinity interactions between enzymes and their cofactors at the electrode interface can allow the efficient electrical contacting of aligned proteins. 

The crystal structure of canine MPO has recently been determined to 3-A resolution (7, 14). This mammalian enzyme is a covalently linked dimer of molecular weight 140 kDa that can be cleaved into two identical halves by reduction of a single disulfide bond. Each half of the dimer, termed hemi-MPO, has the same optical properties and catalytic activity as the parent. Hemi-MPO consists of two polypeptides of 466 and 108 amino acid residues, and a heme-type prosthetic group is covalently bound to the larger polypeptide in a crevice 15 A below the protein surface. Like CCP and LIP, the secondary structure of MPO is dominated by a-helices with relatively little /3-sheet structure (Fig. 

Furthermore, the functionalization of conducting polypyrrole films has been used for formation of surfaces suitable to link enzymes or other proteins covalently [26]. 

The final immobilisation procedure that have been used is cross-linking and involves joining the enzymes to each other forming a large, three-dimensional complex structure, achieved by physical or chemical methods. The chemical methods of cross-linking involve covalent bond formation between the protein molecules by means of a bi- multifunctional res ent such as glutaraldehyde and toluene diisocyanate. ... 

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