Phycobiliproteins conjugation

The algal extract of P. aerugineum is blue, with maximum absorbance at a wavelength of 620 nm and a red fluorescence with maximum emission at 642 nm. The main phycobiliprotein, C-phycocyanin, is the same type of phycocyanin found in most Cyanobacteria. The chromophores are composed of phycocyanobilins, conjugated to an apoprotein via thioether bonds. 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

The SPDP-activated phycobiliprotein may be reacted with a sulfhydryl-containing protein to create a fluorescent conjugate linked through disulfide bonds. 

Other proteins commonly crosslinked to (strept)avidin are chromogenic or fluorescent molecules, such as ferritin or phycobiliproteins (Chapter 9, Section 7). These conjugates can be used in microscopy techniques to stain and localize certain antigens or receptors in cells or tissue sections. 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

Glazer, A.N., and Stryer, L. (1983) Fluorescent tandem phycobiliprotein conjugates Emission wavelength shifting by energy transfer. Biophys. /. 43, 383-386. 

M. N. Kronick and P. D. Grossman, Immunoassay techniques with fluorescent phycobiliprotein conjugates, Clin. Chem. 29, 1582-1586(1983). 

Figure 5-9. Structure of two phycobilins that act as important accessory pigments. Phycoerythrobilin has fewer double bonds in conjugation than phycocyanobilin, so its Xmax occurs at shorter wavelengths (Fig. 5-8). Phycobilins occur covalently bound to proteins that is, they are the chromo-phores for phycobiliproteins.

OiVT, GlazerAN, StryerL (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J Cell Biol 93 981-986... 

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