An improved method for estimating R-phycoerythrin and R-phycocyanin contents from crude aqueous extracts of Porphyra (Bangiales, Rhodophyta)
- 567 Downloads
One frequently-cited method for determining phycoerythrin (PE) and phycocyanin (PC) contents from crude aqueous extracts of red seaweeds utilizes peaks and troughs of absorbance spectra. The trough absorbance values are used to establish a linear or logarithmic baseline attributable to background scatter of particulate cellular debris not removed by centrifugation. Pigment contents are calculated by subtracting baseline values from PE and PC absorbance peaks. The baseline correction is intended to make the method independent of centrifugation time and/or speed. However, when crude extracts of Porphyra were analyzed using this protocol, R-PE and R-PC estimates were significantly affected by centrifugation time, suggesting that the method was not reliable for the genus. The present study has shown that with sufficient centrifugation, background scatter in Porphyra extracts can be removed, the remaining spectrum representing the overlapping absorbance peaks of water-soluble pigments in the extract. Using fourth derivative analysis of Porphyra extract absorbance spectra, peaks corresponding to chlorophyll, R-PE, R-PC, and allophycocyanin (APC) were identified. Dilute solutions of purified R-PE, R-PC and chlorophyll were scanned separately to identify spectral overlaps and develop new equations for phycobilin quantification. The new equations were used to estimate R-PE and R-PC contents of Porphyra extracts and purified R-PE, R-PC and chlorophyll solutions were mixed according to concentrations corresponding to the sample estimates. Absorbances and fourth derivative spectra of the sample extract and purified pigment mixtures were compared and found to coincide. The newly derived equations are more accurate for determining R-PE and R-PC of Porphyra than previously published methods.
Key wordsfourth derivatives phycobilins pigments red seaweeds spectroscopy
- Chl a
- Chl d
This work was funded in part by the USDA New Hampshire Agricultural Experiment Station (NHAES) as Hatch grants #NH00404 and NH00439 and is scientific contribution number 2303 from the NHAES. We would like thank Dr. Leland S. Jahnke, Dr. Arthur C. Mathieson and especially reviewer SB for helpful comments on the manuscript.
- Butler WL, Hopkins DW (1970a) Higher derivative analysis of complex absorption spectra. Photochem Photobiol 12:439–450Google Scholar
- Butler WL, Hopkins DW (1970b) An analysis of fourth derivative spectra. Photochem Photobiol 12:451–456Google Scholar
- Jensen A (1978) Chlorophylls and carotenoids. In: Hellebust JA, Craigie JS (eds) Handbook of phycological methods. Cambridge University Press, Cambridge, pp 59–70Google Scholar
- O’Carra P (1965) Purification and N-terminal analyses of algal biliproteins. Biochem J 94:171–174Google Scholar
- Rowan KS (1989) Photosynthetic pigments of algae. Cambridge University Press, CambridgeGoogle Scholar
- Siegelman HW, Kycia HJ (1978) Algal biliproteins. In: Hellebust JA, Craigie JS (eds) Handbook of phycological methods. Cambridge University Press, Cambridge, pp 71–79Google Scholar