The introduction of phycobiliproteins as fluorescent tags of cells and molecules in 1982 (13) was followed by widespread application of these macromolecules in cell sorting, cell analyses, and immunoassay (5,111. The most commonly used phycobiliproteins are B-and R-phycoerythrins. These proteins have the subunit structure (a/3)Gy with a total molecular weight of -240,000 and contain 34 open chain tetrapyrrole prosthetic groups (bilins) per molecule (3,10,12). Phycoerythrins absorb maximally in the green region of the visible spectrum and emit at 576 nm (2). The (a&y assembly form of phycoerythrin is extremely stable, and no shift in the emission maximum is seen even at 1O-l2M (13).Another phycobiliprotein which has proved valuable as a fluorescent tag is allophycocyanin (AP) (8,9,15). At near neutral pH, this protein exists as a trimer, ( a f i )~, of -100,000 daltons, which contains six bilin prosthetic groups. The trimer has an absorption maximum at 650 nm, an emission maximum at 660 nm, and a quantum yield of -0.4 (2,141. The fact that the excitation and emission maxima for AP lie in the red is particularly important because of the lack of interfering emissions from most biological materials in this region of the spectrum, and because of the ready availability of inexpensive helium-neon lasers. One potential difficulty with AP as a fluorescent tag is that, in contrast to the stability of the phycoerythrins discussed above, AP dissociates at very low concentrations (-1OP8M) to the monomer, a& with a shift in the emission to 645 nm and a decrease in quantum yield. We have recently eliminated this difficulty by preparing a crosslinked AP trimer (XL-AP) which does not dissociate even a t extreme dilution (14). In addition, we have found that XL-AP is markedly more stable at high temperatures than unmodified AP. We anticipate that this AP derivative will be applied widely in cell sorting and analysis. Its properties are such that it may also be well suited as a tag for DNA probes. This report presents data on the wavelength and temperature dependence of the fluorescence of AP and XL-AP. MATERIALS AND METHODS Crosslinked AllophycocyaninAnabaena variabilis AP was purified as previously described (1). To prepare the crosslinked derivative, AP (1.5 mg/ml) in 0.05 M Na-phosphate buffer at pH 7.0 was treated with 35 mM l-ethyl-3-(3-dimethylaminopropy1)carbodiimide (Sigma, St. Louis, MO) for 4 hr at 22°C. The reaction was quenched by the addition of a solution of glycylglycine (Sigma) at pH 7.0 to a final concentration of 0.1 M. The procedure of Ong and Glazer (14) was then used to obtain AP trimer made up of three monoAddress reprint requests to A.N. Glazer,
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