HPLC Separation and Detection

If the holo- and apo-subunits, which might be produced or used as substrates in an enzymatic reaction, can be separated by C4 reverse-phase chromatography as described above, then this method provides an excellent way to detect the transfer of bilin from a holophycobiliprotein to an apo-subunit. Such separations are usually best achieved if the source of the holo-sub-unit is from another organism. The transfer reaction of PCB from Anabaena sp. PCC 7120 holo-a-PC to Synechococcus sp. PCC 7002 apo-a-PC mediated by Syne-chococcus sp. PCC 7002 CpcECpcF was detected using this method (22). Syne-chococcus sp. PCC 7002 CpcECpcF proteins can also transfer a bilin from Syne-chococcus sp. PCC 7002 holo-PC to Anabaena sp. PCC 7120 apo-a-PC sub-

unit (see Figure 6).

4.3.4. Characterization of the Product by Tryptic Digestion

This is the most quantitative method of characterization of the bilin product (2,3,20). The addition product is cleaved using trypsin, and tryptic peptides are separated on a C18 reverse-phase column (45). Tryptic peptides can be collected, their absorption spectra in 10 mM TFA determined, and their composition evaluated by amino acid analysis or sequencing to show rigorously which bilin was added to a particular site(s) on the apophycobiliprotein subunit. If multiple products are present, this is the best method to determine how many products have been formed and to quantitate their relative amounts. Keep in mind that for each phycobiliprotein, digestion by more than one protease may be required to obtain a fragment sufficiently small to allow its isolation and characterization. Digestion procedures for each type of phycobiliprotein have been published (7,49,50,55,67,74-76), and it is recommended that the user refer to the optimized procedure for the particular phyco-biliprotein with which he/she is working. The procedure described below was used successfully on C-PC and R-PE (2,45).

The addition product should be separated from unreacted bilin by chromatogra-phy on Sephadex G-25. The phycobilipro-tein should then be fully denatured by acidification with 1 N HCl to pH 2.0 and stored under N2 for 45 minutes. Trypsin (TCPK-treated; Worthington Biochemical, Lakewood, NJ, USA), dissolved in 1 mM HCl at 5 mg/mL concentration, is added to 2% (wt/wt) to the denatured phyco-biliprotein in HCl. This mixture is titrated to pH 7.5 with 1 N NaOH after the addition of ammonium bicarbonate to 100 mM. After incubation of this mixture for 2 hours at 30°C in the dark, an additional aliquot of trypsin is added, and the incubation is continued for another 2 hours under the same conditions. The reaction is stopped by the addition of glacial acetic acid to 30% (vol/vol). If a large amount of protein is being digested, then fractiona-tion on Sephadex G-50 in 30% acetic acid (vol/vol) is a good method to separate undigested material from tryptic peptides. If the amount of material is scaled for analytical purposes, then the colored material can be collected and loaded directly onto a SepPak C18 cartridge. The cartridge can be washed with 0.1% TFA followed by elu-tion by 60% acetonitrile, 40% 0.1% TFA. The eluate should be collected, dried under N2, and redissolved in 10 mM TFA prior to HPLC separation. However, if the amount of material is scaled for preparative purposes, the colored material in the eluate from the gel exclusion chromatography in 30% acetic acid should then be concentrated under N2 before dilution with 50 mM Na-phosphate, pH 2.5. The mixture should then be fractionated on an ionexchange column (SP-Sephadex G-25, 2 x 6.5 cm) and eluted with a linear gradient of 0 to 0.6 M NaCl in 50 mM Na-phos-phate, pH 2.5. Fractions containing colored material should be collected, desalted on the SepPak C18 cartridge as described above, before separation by HPLC.

The conditions used for separating the tryptic peptides of phycocyanin follow. However, for each phycobiliprotein, different gradient conditions may be required, and optimization of these conditions should be pursued prior to preparative-scale analyses. For the phycocyanin of Synechococcus sp. PCC 7002, a C18 reversephase analytical column (5 pm, 4.6 x 250 mm) should be used for separation of tryptic peptides (see Figure 7). The solvent system is 0.1 M Na-phosphate, pH 2.1 (Buffer A) and acetonitrile (Buffer B) with flow rates of 1.5/mL min. Peptides are loaded at 20% Buffer B (80% Buffer A)

Figure 6. Monitoring the transfer of bilin from Synechococcus sp. PCC 7002 PC to Anabaena sp. PCC 7120 apo-a-PC by C4 reverse-phase HPLC. Each assay contained 100 pg of Anabaena sp. PCC 7120 apo-a-PC, 75 pg of Synechococcus sp. PCC 7002 PC, 0.2 pM Synechococcus sp. PCC 7002 CpcECpcF (if present) in a volume of 400 pL (reaction assay buffer conditions are as described in Figure 5). Reactions were allowed to proceed for 16 hours at room temperature in the dark. Each reaction was combined with 800 pL of 9 M urea, pH 1.9, mixed, and centrifuged prior to injection on the C4 column (as described in this chapter). After injection, buffer conditions (buffers are those from Swanson and Glazer; Reference 66) are as follows: 2 minutes at 35% Buffer B (65% Buffer A), a 1-minute linear gradient to 53% Buffer B (47% Buffer A), followed by a linear gradient to 63% Buffer B over 20 minutes (22). Each assay was monitored at 280 nm (reflecting protein content) and 680 nm (reflecting bilin content). Retention times for various components are as follows: Anabaena sp. PCC 7120 apo-a-PC, 9.5 minutes; Anabaena sp. PCC 7120 holo-a-PC, 10 minutes; Synechococcus sp. PCC 7002 apo-a-PC, 11.7 minutes; Synechococcus sp. PCC 7002 holo-a-PC, 12.2 minutes; Synechococcus sp. PCC 7002 holoP-PC, 15.8 minutes. Synechococcus sp. PCC 7002 CpcECpcF is capable of transferring bilin from 7002 holo-a-PC to Anabaena sp. PCC 7120 apo-a-PC (W.M. Schluchter and A.N. Glazer, unpublished results).

Figure 6. Monitoring the transfer of bilin from Synechococcus sp. PCC 7002 PC to Anabaena sp. PCC 7120 apo-a-PC by C4 reverse-phase HPLC. Each assay contained 100 pg of Anabaena sp. PCC 7120 apo-a-PC, 75 pg of Synechococcus sp. PCC 7002 PC, 0.2 pM Synechococcus sp. PCC 7002 CpcECpcF (if present) in a volume of 400 pL (reaction assay buffer conditions are as described in Figure 5). Reactions were allowed to proceed for 16 hours at room temperature in the dark. Each reaction was combined with 800 pL of 9 M urea, pH 1.9, mixed, and centrifuged prior to injection on the C4 column (as described in this chapter). After injection, buffer conditions (buffers are those from Swanson and Glazer; Reference 66) are as follows: 2 minutes at 35% Buffer B (65% Buffer A), a 1-minute linear gradient to 53% Buffer B (47% Buffer A), followed by a linear gradient to 63% Buffer B over 20 minutes (22). Each assay was monitored at 280 nm (reflecting protein content) and 680 nm (reflecting bilin content). Retention times for various components are as follows: Anabaena sp. PCC 7120 apo-a-PC, 9.5 minutes; Anabaena sp. PCC 7120 holo-a-PC, 10 minutes; Synechococcus sp. PCC 7002 apo-a-PC, 11.7 minutes; Synechococcus sp. PCC 7002 holo-a-PC, 12.2 minutes; Synechococcus sp. PCC 7002 holoP-PC, 15.8 minutes. Synechococcus sp. PCC 7002 CpcECpcF is capable of transferring bilin from 7002 holo-a-PC to Anabaena sp. PCC 7120 apo-a-PC (W.M. Schluchter and A.N. Glazer, unpublished results).

and eluted with a linear gradient to 40% Buffer B (60% Buffer A) over 20 minutes (2).

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