Enzyme bridge technique

Using a combination of techniques such as EPR, resonance Raman, and MCD spectroscopy, the conversion of [2Fe-2S] into [4Fe—4S] centers has been found to take place under reducing conditions in E. coli biotin synthase 281). The as-prepared form of this enzyme has been thought to contain one [2Fe-2S] center per monomer, coordinated by the three cysteine residues of the motif Cys-X3-Cys-X2-Cys and by a fourth, noncysteinyl ligand. Upon reduction, a [4Fe-4S] cluster bridging two monomers may be formed in the active enzyme. In the reduced state, the [4Fe-4S] center is characterized by a mixture of S = I and S = k spin states giving EPR features at g 5.6 and... 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

Positional isotope exchange ( PIX ) is a very valuable technique in determining enzyme mechanisms, particularly those utilizing ATP, GTP, or another NTP substrate -. For example, the nucleotide substrate can be labeled with in bridging (i.e., P—O—P or phos-phoanhydride oxygen) and/or its nonbridging positions. 

When 31P is bonded to lsO the chemical shift of the 31P is altered by 0.0206 ppm from that when the phosphorus is bonded to leO. This allows lsO labels introduced into phospho groups to serve as tracers which can be followed continuously during reactions.683 The technique is useful in studies of stereochemistry (see Section 2) and for examination of positional isotope exchange.690 This latter technique is often used with ATP containing lsO in the P,y-bridge position. If an enzyme transfers the terminal (y) phospho group to an acceptor via a phosphoenzyme but without loss of the ADP, we may expect positional isomerization. The lsO will move between the P,y-bridge position and... 

Biotinylated enzymes can be used as detection reagents in avidin—biotin assay procedures. Particularly, in the bridged avidin—biotin (BRAB) approach or the ABC technique (Chapter 13, Section 2), a biotin-labeled enzyme is used as the signaling agent... 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

This latter technique is often used with ATP containing 0 in the P,y-bridge position. If an enzyme transfers the terminal (y) phospho group to an acceptor via a phosphoenzyme but without loss of the ADP, we may expect positional isomerization. The 0 will move between the p,y-bridge position and... 

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