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,

The specific binding of ³H-estradiol by the nuclear fraction of the hypothalamus and anterior pituitary in male and female immature rats was examined. Anterior pituitaries and hypothalami were incubated in vitro with either ³H-estradiol or ³H-estradiol plus diethylstilbestrol, and the amount of ³H-estradiol in the nuclear fraction was determined. This procedure permits an evaluation of the concentration of specific estrogen binding sites, or the receptor estradiol complex (R · E), present in the nuclear fraction, and the dissociation constant, K^R_d, of the R · E complex. No significant differences were observed in the concentration or K^R_d of R · E when male and female pituitaries were compared. Nuclear R · E complexes in the female hypothalamus were measurable and appeared to be similar to those of the pituitary with respect to K^R_d however, the concentration of R · E was much lower. No nuclear R · E was observed in the male hypothalamus or in a control CNS tissue, female cerebral cortex. It is suggested that testosterone secretion in the developing male rat may alter either the concentration of hypothalamic R · E or its ability to be translocated to the nucleus, while it has little or no effect on the estrogen receptor in the pituitary.

CP, Calc'd 7.4; found 7.5. " van Slyke wet carbon. h S, Calc'd 7.4; found 7.5. { CP, Calc'd 11.4; found (Volhard) 11.3. ' Melting points were taken either in a bath or on a Fisher-Johns block. k II ratio = µ g. histamine (to produce a given contraction)/^g. compound (required to suppress this response), and was determined on the isolated guinea pig gut by the method of ( 14).1 For examples of the various methods, see Experimental.

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