Surface labeling reagents

In recent years there has been considerable interest in the structure of biological membranes, and photochemical reagents capable of yielding low resolution structural information about membrane proteins have been developed. Several examples of photochemical surface-labeling reagents have appeared, and much effort has been devoted to the development of photoactivatable hydrophobic reagents for labeling from within the lipid bilayer. 

Photochemical surface labeling reagents introduced by Staros and Richards (1974) have a number of potential advantages. First, the high 

More recently it has become clear that NAP-taurine passes through other membranes more readily than those of erythrocytes. It has proved difficult, for instance, to prevent its penetration into other types of mammalian cells. This limits its use for labeling membranes from the external medium. An even more important problem is that the molecule appears to bind to cell membranes like a detergent (Dockter, 1979 Richards and Brunner, 1980) and it seems that a number of the minor proteins labeled in intact erythrocytes penetrate the membrane deeply from the cytoplasmic side but do not span it. These proteins are accessible to NAP-taurine molecules that reach down into the hydrocarbon region of the bilayer. 

A special feature of the Dockter s reagent is that the reaction products are fluorescent although the sensitivity of detection possible with a radio-labeled reagent cannot be achieved in most laboratories.  

A macromolecular, photochemical surface labeling reagent was developed by Louvard et al. (1976). It comprised a Fab fragment of a human myeloma protein with which 4-fluoro-3-nitrophenylazide (Table 5.1.1c) had been reacted. Used at a concentration of 0.1 mM, this conjugate could be used to label the surfaces of sealed vesicles or it could be trapped inside vesicles and used to label from within. Louvard and colleagues focused their attention on the modification of aminopeptidase in intact brush-border membranes and demonstrated that the enzyme spans the bilayer. From the 

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Photoactivatable, hydrophobic reagents for membranes were developed during the same period as photoactivatable, surface labeling reagents, but... 

Aryl azides fluorescent hydrophobic and surface labeling reagents... 

DETECTION OF INTERMEDIATES BY USE OF NONSPEOnC SURFACE-LABELING REAGENTS... 

In another case in which a number of exemplary control experiments were done, Markwell and Fox (1980) crosslinked the outer membranes of enveloped viruses with methyl 3-(p-azidophenyl)dithio]propionimidate. Virus (4 mg protein/ml) was reacted with the imidate (0.1 to 0.5 mM) at 0°C for 30 min at pH 8.5. The reaction was quenched with 50 mM ammonium acetate, 50 mM NEM (30 min, 25 °C), and the vims recovered by centrifugation. After irradiation the crosslinked polypeptides were examined in a two-dimensional SDS-polyacrylamide gel. One complication was that the crosslinking pattern had to be compared with a native pattern of disulfide linkages, and a reagent with a different cleavable crosslink may have been a better choice. As mentioned above, the analysis was simplified by the use of surface labeling. 

Live worms incubated with, 26l-labelled reagents (Chloramine T, Bolton-Hunter, lodogen) to label proteins on surface... 

The substrate is cleaved by the enzyme-labeled hapten in the immunocomplex at the surface. The fluorescence can be measured in solution or at the surface. For the determination of high molecular mass antigens a two-site sandwich assay can be applied. A variation represents the double antibody assay, whereby the second antibody, which is directed against the hapten-specific antibody, is enzyme-labeled. Reagent-excess based assays are mainly used for the determination of antibodies in a noncompetitive format. In Scheme 6, a procedure for an indirect ELISA for the determination of antibodies is described. 

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