Deborah Guard-Friar

The Chromophore and Polypeptide Composition of Aplysia Ink

The composition of the ink of the sea hare, Aplysia, was studied in regard to its tetrapyrrole and polypeptide content. The ink was separated into three pigment components by both thin-layer and gel filtration chromatography. These three pigments have distinctive visible absorption spectra, and--by comparison with known tetrapyrroles--we have demonstrated that they are derived from the three tetrapyrrole chromophores (bilins) found on the biliproteins of certain red algae, which constitute a portion of the Aplysia diet. The red component is phycourobilin; the purple is phycoerythrobilin; and the blue is phycocyanobilin. Sodium dodecyl sulfate gel electrophoresis experiments were also performed. The results of these experiments showed several polypeptides, and major bands at 78,000 and 61,000 molecular weight were noted. Biliproteins, at most, would be minor components of the ink.

Picosecond fluorescence spectroscopy of the biliprotein phycocyanin 612: Direct evidence for fast energy transfer

Abstract Energy-transfer processes in phycocyanin 612, a light-harvesting biliprotein from the cryptomonad alga Hemilselmis virescens , have been studied by picosecond fluorescence spectroscopy. Fluorescence was excited with a 30 ps 532 nm laser pulse and detected with a streak camera. Two types of data were obtained. Profiles of intensity versus time using 10 nm band pass interference filters are summarized as follows: emission at wavelengths 650 nm) exhibits a 7 to 10 ps exponential rise time and a long exponential decay (1.0–1.4 ns). In the region from 600 to 640 nm, the time-resolved emission is more complicated; it consists of at least the sum of the shorter and longer wavelength components but additional components cannot be ruled out. The second type of data consists of spectra (intensity versus wavelength) taken at various times relative to the excitation pulse. These spectra qualitatively confirm the interference filter results. The kinetic results are interpreted in terms of Forster energy transfer between spectrally different chromophores within the biliprotein. Thus the emission of phycocyanin 612 provides a direct probe for studying the fast component of fluorescence during an energy transfer process.

SUBUNIT SEPARATION (α, αprime;, β) OF CRYPTOMONAD BILIPROTEINS *

Abstract— Phycocyanin 645, phycocyanin 612 and phycoerythrin 545 are biliproteins isolated from the cryptomonad algae, Chroornonas species, Hemiselmis virescens and Rhodomonas lens, respectively. The protein has α and β subunits. which are separated on an ion exchange column by using a urea gradient and omitting 2‐mercaptoethanol from the solvent. This separation establishes that the α and β subunits are not joined by disulfide bonds. In addition it has recently been shown that mercaptoethanol can produce spurious results in the calculation of the chromophore contents of these biliproteins (Guard‐Friar and MacColl, 1984). The mercaptoethanol‐free experiments allow analysis of the chromophore content in a rapid and artifact‐free manner. When the ion exchange chromatography of phycocyanin 645 is manipulated by changing the type of urea gradient, two distinct a subunit fractions are obtained. These two fractions have identical visible absorption spectra but different amino acid compositions. At least two different gene products are, thus. responsible for the a subunits. The sole chromophore on the two a subunits of phycocyanin 645 is the unique 697‐nm bilin as seen in acidic urea. Its reactivity with mercaptoethanol is determined. The α subunits of phycoerythrin 545 have two different bilins: cryptoviolin and phycoerythrobilin. Phycocyanobilin is the chromophore on the α subunit of phycocyanin 612.

The development of exciton migration routes for phycocyanin 645 and allophycocyanin.

Abstract— The origin of the comparatively complex absorption spectrum of the cryptomonad biliprotein, phycocyanin 645 from Chroomonas species, has been investigated by deconvolution of its absorption and CD spectra together with fluorescence polarization studies. The visible absorption and CD spectra were each deconvoluted into four components, three pure Gaussian and one Gaussian‐Lorentzian chimera. The difference spectrum between the visible absorption spectra of partially renatured and partially dissociated protein and the fluorescence polarization spectrum are compared to these deconvolutions. All results are consistent with a previous proposal that band splitting from a pair of strongly‐coupled dipoles contributes to the absorption and CD spectra of this biliprotein. A model for the flow of exciton migration through this protein is presented that incorporates these data [together with appropriate literature reports]. This exciton migration model together with one for the biliprotein, allophycocyanin, includes both strong and very weak coupling of dipoles. This combination of mechanisms has salient influence on the visible absorption spectra and the routes of exciton migration characteristic of these two proteins.

Chromatographic and spectroscopic analysis of phycoerythrin 545 and its subunits

Phycoerythrin 545 is a light-harvesting biliprotein isolated from the cryptomonad Rhodomonas lens. Although the absorption spectrum of the native protein suggests that this protein has only phycoerythrobilins for its chromophores, the denaturated protein shows a small near ultraviolet absorption band with a maximum at 333 nm which is not present in phycoerythrobilin. Two methods were employed to separate the α and β subunits of this protein: chromatography with Sephacryl S-200 in acidic urea or centrifugation in a sucrose density gradient at pH 3.0. The chromatography experiments yielded two bands, which were shown to be pure α or β subunits by sodium dodecyl sulfate gel electrophoresis. The absorption spectrum of β showed only phycoerythrobilins, but the spectrum of α was not like that of any known bilin chromophore. Its absorption spectrum could be constructed by a combination of phycoerythrobilin and cryptoviolin. The β subunit separated on the sucrose density gradient was highly aggregated. Circular dichroism and fluorescence polarization spectroscopy indicated that this aggregated β subunit has chromophores in atypical environments. Comparison of the absorption, spectra of native and denatured phycoerythrin 545 suggests that chromophores in the native state are held by the protein in a more linear confirmation.

The route of exciton migration in phycocyanin 612

Abstract The biliprotein, phycocyanin 612, was purified from the cryptomonad, Hemiselmis virescens , and its chromophore content confirmed by an analysis of its tryptic peptides. Chromatography of a tryptic digest indicated that the isolated protein has three different phycocyanobilin chromopeptides and a single cryptoviolin chromopeptide. Spectroscopic data indicated that each different chromopeptide occurs twice for a total of eight chromophores on each oligomer. The tryptic digest data and the results from earlier experiments suggest that the protein may have two identical sets of four chromophores that comprise an energy-transfer unit within the protein. CD spectroscopy on phycocyanin 612 in the visible region of the spectrum was used to obtain information on the topography of these chromophores. Two chemicals, potassium permanganate and sodium thiocyanate, were used to alter the CD spectrum. The effects of both these chemicals suggested a close relationship between two of the four observed components in the visible CD spectrum of this biliprotein. This chromophore relationship is assigned to strong coupling of the dipoles of the two lowest-energy chromophores and produce exciton delocalization between these chromophores. Each dimeric protein has two identical delocalized pairs that produce spectral splitting and are the fluorescence emitters of the isolated protein. Based on this evidence and data from the literature, a tentative model is presented that shows the exciton migration route for phycocyanin 612. After excitation of the highest-energy chromophore, cryptoviolin, excitons are transferred by very weak dipole coupling to the highest-energy phycocyanobilin and from there by the same mechanism to the strongly coupled pair of phycocyanobilins. The identical process occurs on both halves of the protein.

Excitation energy transfer between sensitizing chromophores of phycocyanin 612

Abstract. The visible absorption and circular dichroism spectra of phycocyanin 612 were each resolved into four components. The deconvolution required the blue side of each spectrum to be fitted by a half‐Lorentzian band and the remainder of the spectrum by Gaussian components. The energy levels of the components in the deconvolution allowed analysis of the discrete steps in the transfer of excitation energy within the biliprotein. By comparison of the deconvolution of the absorption spectrum to the published results from ultrafast fluorescence kinetics of phycocyanin 612, the times for s‐to‐s and s‐to‐f excitation energy transfer are established. This is the first evidence of excitation energy transfer between two types of sensitizing (s) chromophores in a biliprotein. The analysis of the ultrafast kinetics and the deconvolution of the absorption spectrum allowed the assignments of 7–10 ps to the transfer of excitons from an s to a fluorescing (f) chromophore and a time faster than 7 ps to the transfer of excitons between two spectrally distinct s chromophores. The steady‐state fluorescence polarization spectrum of this biliprotein supports the hypothesis that excitation energy transfer occurs between s chromophores.

Phycoerythrin 566 — a fluorescence study

The cryptomonad biliprotein, phycoerythrin 566 from Cryptomonas ovata , has been studied by a variety of spectroscopic technics, including absorption, fluorescence, fluorescence polarization, and ultrafast fluorescence kinetics. The data were analyzed to obtain information about the transfer of excitons among the various chromophores (bilins) on the isolated protein. Ultrafast fluorescence studies were performed using a variety of interference filters to monitor emission after excitation at 532 nm. The fluorescence kinetics was found to be dependent on the emission wavelength being observed. The kinetic fits of the data suggest that 532-nm light excited two typesof chromophores and that one type transferred energy to the other in about 30 ± 3 ps. This conclusion was based on the fitting of the fluorescence to two kinetic equations in which the rate of energy transfer out of one chromophore was equalto the rate of energy transfer into another chromophore. Emission from the protein occurred with a 1000-ps radiative lifetime. The fluorescence polarization spectrum of phycoerythrin 566 was consistent with several energy-transfer events between various energetically different chromophores. The various steady-state fluorescence results suggested that one type of chromophore, or a group of chromophores in equilibrium, was the final emitter. After absorption of photons, excitons were transferred with great efficiency (97%) between various spectroscopically distinct chromophores and were eventually emitted by the lowest-energy chromophores. These results were for a dimeric (α 2 β 2 ) protein at pH 6.0; at lower pH or lower protein concentration, the protein dissociated.