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aExtinction coefficients are mM-1cm-1 at the wavelength indicated. The absorption spectra of peptide-linked bilins were measured in 10 mM TFA or 8 M urea, pH 1.9. These values are taken from References 7, 33, and 44.

chococcus sp. PCC 6301 and Synechocystis sp.; (22663; ATCC, Manassas, VA, USA) also called Microcystis aeruginosa (14) (Procedure 5).

❖ Procedure 5. Separation of Allophycocyanin Subunits from Synechococcus sp. PCC 6301 and Synechocystis sp.

1. Dissolve 325 mg purified and lyo-philized AP in 50 mL of 10 mM K-phosphate, 8 M urea, 10 mM P-mer-captoethanol, pH 8.0, and equilibrate for 1 hour at room temperature. See section 2.1.

2. Load the material onto a DEAE Sephadex A-50 column (3.5 x 12 cm) and wash with equilibration buffer.

3. Use 200 mL of equilibration buffer plus 50 mM KCl to elute the elute the P subunit of AP

4. Residual P subunit is eluted by repeated washes with 150 mL equilibration buffer plus 80 mM KCl.

5. Use equilibration buffer plus 180 mM KCl to elute the a subunit of AP

6. Pool fractions of each subunit from steps 3 and 5 for dialysis against 25 mM ammonium acetate, pH 6.8, and concentration by ultrafiltration using an Amicon® cell with a 10 000 MWCO membrane (Millipore, Bedford, MA, USA).

A method similar to the one developed for the separation of the PC subunits was successfully used in the separation of the a, P, and y subunits of phycoerythrin from P cruentum (34). The only significant difference was in the development of the column. The y subunit was eluted with 7.4 M urea, the a subunit with 8.0 M urea, and the P subunit with 9.0 M urea. Similar conditions were used to separate the sub-units of phycoerythrin II (PE II) from the cyanobacterium Gloeobacter violaceus (10).

The Bio-Rex 70 column (1.5 x 15 cm) with the PE subunits adsorbed was washed with 15 mL of 2.0 M urea, 30 mL of 4.0 M urea, and 50 mL of 6.0 M urea before development with a linear gradient of 6.0 to 10.0 M urea, pH 3.0 (20 mL total volume). The a subunit eluted first followed by the P subunit. Subunits were renatured by exhaustive dialysis against 50 mM K-phosphate buffer at pH 7.0 at room temperature.

Separation of the subunits of Anabaena variabilis PEC was first demonstrated by Bryant et al. using a modification of the method developed for the separation of PC subunits described above (12). The BioRex 70 column (3.9 x 51 cm) was subjected to incremental step gradients of acidic urea as described previously, followed by elution of the a subunit by addition of 7.4 M urea, pH 3.0. Once the elution of the a subunit was complete, elution of the P subunit was accomplished by addition of 9.0 M urea, pH 3.0. Subunits were dia-lyzed against 50 mM ammonium acetate, pH 6.8.

2.5. Purification of Phycobiliproteins by HPLC

In 1987, HPLC was used to verify the purity of PC and AP preparations from M. aeruginosa (58); however, the method also showed that the AP and PC subunits could be separated on a C^ reverse-phase column. In 1990, Swanson and Glazer introduced a method for separation of phycobiliprotein subunits using C4 reverse-phase HPLC (66). These HPLC methods have several advantages over the conventional chromatographic methods. They are more rapid and require much less starting material. When used in conjunction with a photodiode array detector, these methods also give immediate spectroscopic information about bilin content and subunit stoichiom-etry. The method of Swanson and Glazer has also successfully been used to separate phycobiliproteins obtained directly from purified phycobilisomes, giving quantitative information regarding phycobilipro-tein stoichiometry and content in these mixtures (39). When both HPLC methods were compared, the method of Swanson and Glazer gave better resolution of phycobiliproteins isolated from Arthrospira maxima (38,39). The use of reverse-phase HPLC is clearly a better choice than conventional chromatographic procedures for determining stoichiometric information when the amount of starting material and speed are primary concerns.

The method of Swanson and Glazer uses a C4 reverse-phase analytical column (250 x 10 mm) and a solvent system consisting of 0.1% TFA in water (Buffer A) and a 2:1 acetonitrile: isopropanol mixture (Buffer B). This purification procedure has been very successful in the separation and resolution of diverse types and mixtures of phycobiliproteins. The purified phyco-biliprotein or phycobiliprotein mixture, typically 100 to 1500 ^g in 200 to 500 ^L in 5 mM Na-phosphate, pH 7.0, 1 mM P-mercaptoethanol is combined with an equal volume of 9.0 M urea, pH 2.0 (freshly prepared), and subjected to centrifuga-

Figure 3. HPLC separation of cyanobacterial C-PC subunits. Purified PC from Synechococcus sp. PCC 6301 (top panel) or Anabaena sp. PCC 7120 (bottom panel) was separated on a C4 reverse-phase HPLC column as described in the text. Elution of subunits was monitored at 660 nm in order to follow the absorbance of peptide-linked PCB. In each case, the a subunit elutes prior to the ß subunit. This figure was modified with permission from Reference 66.

tion in a microcentrifuge for 5 minutes prior to injection on the column. A Hi-Pore® RP304 column (Bio-Rad Laboratories) equilibrated in 65% Buffer A and 35% Buffer B (1.5 mL/min) has typically been used. After injection of the sample, proteins are eluted with a linear gradient to 30% Buffer A and 70% Buffer B over 35 to 40 minutes depending upon the source of the phycobiliprotein (see Figure 3). With a few alterations of the elution gradient profile, this method has been successfully employed in the separation of a wide variety of phycobiliproteins from cyano-bacteria, red algae, and cryptomonads (17,29,39,66,74). In fact, researchers have had success in the separation of phyco-biliproteins from phycobilisome samples taken directly from sucrose gradients (after dialysis against 5 mM Na-phosphate, pH 7.0 followed by combination with an equal volume of 9.0 M urea, pH 1.9, prior to injection) (see Figure 4). Toole et al. combined the phycobilisomes taken directly from sucrose gradients (in sucrose-phosphate) with an equal volume of 8.4 M guanidine hydrochloride, pH 6.4 (followed by centrifugation), prior to loading on the C4 column (Vydac/The Separations Group, Hesperia, CA, USA) using the gradient conditions described above (72). This method has also been successfully used to characterize the linker polypeptide and phycobiliprotein stoichiometry in phyco-bilisomes from A. maxima (38,39).

Some technical considerations to keep in mind for each separation include the need to use higher concentrations of urea to solubilize phycobiliprotein mixtures that may contain any given apophycobilipro-tein. It has been observed that apophyco-biliprotein subunits often do not bind as well as holo-subunits under these conditions, but that addition of urea to at least 6 M final concentration in the solution to be injected greatly increases the yield of apophycobiliprotein material (22). It is also very important to wash the column extensively between injections using a linear gradient to 100% Buffer B over 5 minutes followed by at least 5 to 10 minutes of washing the column with 100% Buffer B. The P subunits of phycobiliproteins are sometimes retained on the column, and these will usually be eluted by this treatment. If careful quantitation of a sample is required, it is wise to perform a blank injection between each run with samples in order to insure that the column is entirely free of residual phycobiliprotein subunits.

Preparative separation of phycobilipro-tein subunits can be accomplished using this method in conjunction with a semi-preparative C4 reverse-phase column (or by employing multiple runs on an analytical column). Subunits can be collected as they elute from the column, and the solvents can be removed by rotary evaporation. The aqueous subunits can then be diluted 2:9 with 9.0 M urea, pH 2.0, 10 mM P-mer-captoethanol, followed by dialysis against 50 mM Na-phosphate, pH 7.0 (22).

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