Evaluation of methods for the separation and analysis of proteins and free amino acids in phytoplankton samples

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Abstract
Due to their importance as not only major constituents in paniculate matter but also the metabolism of nitrogen in marine microorganisms, numerous methods have been employed to measure proteins and free amino acids. However, two difficulties frequently complicate these measurements. First, an initial separation of proteins from free amino acids is helpful since most analytical methods are somewhat sensitive to both compound types. Second, the choice of detection techniques that minimize response differences between various proteins or amino acids is desirable since natural samples of microorganisms consist of mixtures of many proteins and amino acids. To address these problems, four protein detection techniques (modified Lowry et al., Dorsey et al., Bradford and fluorescamine) and two amino acid detection techniques (fluorescamine and o-phthaldialdehyde) were evaluated. Relative extraction efficiencies for protein from phytoplankton samples were also evaluated with six homogenization solutions/protocols (TCA, NaOH, boiling NaOH, Triton X-100, NaOH plus Triton X-100 and distilled water). TCA homogenization yielded the highest protein recoveries, and sufficient physical separations between proteins and free amino acids were obtained with TCA concentrations between 0.18 and 0.37 M. Results of these studies allowed for development of a method for extracting, separating and analyzing proteins and total free amino acids from a common phytoplankton sample. The procedure involves initial homogenization in a TCA solution, followed by centrifugation to separate protein and free amino acid fractions. Proteins are then analyzed by a modification of the Lowry et al. procedure, and amino acids by a fluorescamine procedure.

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