Determination of protein for studies of marine herbivory: a comparison of methods

In this study we compared techniques for the estimation of protein for studies of marine herbivory. As an example we use gut fluid from the temperate marine herbivorous fish Kyphosus sydneyanus. There were two components to this work: (1) to compare the ability of spectrophotometric methods to estimate protein in gut fluid; and (2) to determine the effect of freezing digesta samples on the extraction of gut fluid for the measurement of total hydrolysable amino acids (THAA). The Bradford, Lowry, deoxycholate/trichloroacetic acid (DOC/TCA) Lowry and the bicinchoninic acid (BCA) spectrophotometric protein assays were compared for their ability to estimate protein in pooled gut fluid and compared with protein determined by quantitative amino acid analysis using an automated procedure. The spectrophotometric assays all gave differing estimates of protein content and none correlated well with quantitative amino acid analysis. The Lowry and BCA methods gave similar estimates of protein, which were higher than those measured in the same samples by amino acid analysis. Both the DOC/TCA Lowry and the Bradford assay substantially underestimated protein in pooled gut fluid. Internal standard recovery experiments for the protein assays showed interference of colour formation in the Bradford method but not the other spectrophotometric protein assays. This suggested the low values in the Bradford assay were due to gut fluid interfering with colour formation. Therefore, five algal species known to occur in the diet of Kyphosus sydneyanus were selected to test for their interference in the Bradford assay. The algae included three species of phaeophytes, one rhodophyte and one chlorophyte. Extracts from all five algal species interfered with this protein assay to significant but varying degrees. Two methods of extracting gut fluid were compared: (i) method A, which involved removing the gut fluid from the digesta immediately post capture; and (ii) method B, in which samples of digesta were frozen and gut fluid removed later. The fluid from method B gave significantly higher levels of THAA in some gut segments. Freeze–thawing of digesta in method B was thought to be the basis of the increased THAA. This finding suggests that method A more accurately reflects in vivo gut fluid THAA concentrations. The above methodology provides a framework to compare the ability of different herbivorous species at extracting THAA from their diet. These results highlight that the estimation of