Chromophore-labeled proteins

Experience has shown that the chromophore label must be chosen with regarding to the properties of the protein to be dyed and the purpose of the labeling. Among the main properties of the proteins to be labeled, the number and character of the interacting sites with the labeling agent must first be considered. A few examples... 

Labeled proteins are used in enzymology especially as substrates for determining the activity of proteolytic enzymes, for studying structural properties of enzyme molecules (7.2), and for some analytical purposes. Here, attention is paid to the determination of proteolytic activities when proteins labeled with radionuclides, chromophores and fluorophores are used as substrates. 

Unlike bovine serum amine oxidase, which is available in gram quantities and was used as the prototypic system for the establishment of TPQ, a typical preparation of LO yields about 5 mg (starting with about 500 grams of aorta tissue). In an effort to minimize protein loss, purification was stopped after the stage where LO (32 kDa) coelutes with a second protein (24 kDa) from Sephacryl S-200 gel filtration. The two-banded protein was labeled with [ 4C]phenyl-hydrazine, to yield the expected chromophore for a phenylhydrazone derivative of a quinone structure (Fig. 1). Analysis of the i C-labeled protein by SDS gel electrophoresis showed almost exclusive incoiporation of into the 32-kDa LO band (13). Therefore, the unreactive 24-kDa band would not affect subsequent procedures since selection of active site-derived peptides from LO was dependent on screening for in addition to monitoring die UV-Vis absorption of the newly formed chromophore. 

Fig. 10.3-23 A general strategy for the of unmodified protein via filtration. The purification of chromophore-labeled captured proteins can be eluted from the...

The difficulty of the fluorescence detector for the cIEF is the lack of a chromophore in most proteins in the visible wavelength region. Labeling proteins with fluorescence dyes may change their pi values. In addition, multiple labeling is unavoidable for high sensitive detection. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

He X, Bell AF, Tonge PJ (2002) Isotopic labeling and normal-mode analysis of a model green fluorescent protein chromophore. J Phys Chem B 106 6056-6066... 

The general strategy for establishing the sites of protein-chromophore interactions in rhodopsin involves introduction of selective 13 C labels at each position along the length of the retinal chromophore. 

L. Strekowski, M. Lipowska and G. Patonay, Substitution reactions of a nucleofugal group in heptamethine cyanine dyes. Synthesis of an isothiocyanato derivative for labeling of proteins with a near-infrared chromophore, J. Org. Chem. 57.4578-4580(1992). 

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