FIGURE 2.
Isolation of functional PC-APC complexes. A, separation of cross-linked PC-APC products by HPLC-anion exchange chromatography (protocol 2). The arrow indicates the elution position of a single fraction that exhibited energy transfer to APC (λex = 540 nm). B, absorption and fluorescence of the PBS before and after cross-linking and isolation. PBS absorption in 0.9 m phosphate buffer (black dashed line), fluorescence emission of isolated non-cross-linked PBS in low ionic strength buffer (solid gray line), and in high phosphate buffer (gray dashed line). The absorption (black solid line) and fluorescence emission (black dotted line) spectra of the isolated cross-linked PC-APC fractions in low ionic strength buffer are also shown. In all fluorescence measurements, λex = 540 nm. The cross-linked adduct presents a PBS-like absorption curve with the contributions of PC at 620 nm and APC at 620 and 650 nm. PC transfers energy to APC is indicated by the strong emission at >660 nm in the cross-linked sample.