Evaluation of phycobilins in marine cryptomonad rhodomonas salina and marine cyanobacteria synechococcus spp.

Abstract

Phycobiliproteins, present in Cyanophyta, Rhodophyta and Cryptophyta, harvest green, orange, and red regions of the spectrum where chlorophylls and carotenoids absorb poorly. Phycobiliprotins are not like chlorophylls and carotenoids that are extracted with acetone or methanol; therefore, they are difficult to analyze with methods routinely used for chlorophylls and carotenoids. The long-term goal of this work was to develop procedures for the rapid extraction and analyses, both quantitative and qualitative, of phycobilins that could be used in laboratory-grown cultures and more importantly from field-collected samples. Two model algae with well characterized phycobiliproteins were studied: Rhodomonas salina (cryptophyte) and Synechococus spp. RS9917 (cyanophyta). Glycerol-induced uncoupling of phycobiliproteins was studied using in vivo fluorescence assays of R. salina under two growth conditions; results with R. salina were surprisingly similar with published results thought to be unique to Synechococcus, suggesting this may be a new approach to study phycobiliproteins in cryptophytes. For phycobiliprotein isolation studies, cells were harvested by centrifugation and the cell pellets extracted with four 0.1 M buffers (Tris-HCl buffer; acetate buffer; sodium phosphate buffer; and potassium phosphate buffer) at different pH values (1-9). Pellets were disrupted by three cycles of freeze-thaw-sonicate, followed by centrifugation; supernatants were analyzed by absorption spectrophotometry. Co-extraction of phycobiliproteins and chlorophylls/carotenoids was problematic, especially at pH > 6; hence, cells were pre-extracted in 90% acetone to remove chlorophylls/carotenoids. Phycobiliprotein extraction was tested with different buffers/pH combinations, with and without added SDS and to a lesser extent, different concentrations of urea. SDS enhanced extraction of phycobiliproteins from cells pre-extracted in acetone, frequently yielding complete extraction. SDS resulted in altered spectral properties of phycobiliproteins in some, but not all cases. Treatment of SDS extracts with 8M acidic urea (pH 2) or 20% acetic acid resulted in bilin-protein spectra that were largely in line with published spectra; this observation indicates that inclusion of SDS as part of the extraction buffer does not irreversibly affect the bilins during isolation from cells and that the ‘native’ bilin spectra are recovered following dilution in acidic urea, perhaps amenable to quantitative analyses using published extinction coefficients. Phycobiliproteins were also analyzed both quantitatively and qualitatively by HPLC, using protease digested whole cell extracts; results demonstrated resolution of discrete bilin-peptide conjugates. Given the very high protein concentrations present in crude cell extracts, it is likely that protease treatments need to include very high enzyme concentrations and/or much longer digestion periods.