Journal of Applied Phycology (2005) 17: 447–460 DOI: 10.1007/s10811-005-1641-4
C Springer 2005
An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae ∗
Elisabete Barbarino1,2 & Sergio O. Louren¸co2, 1
Programa de P´os-Graduac¸a˜ o em Biotecnologia Vegetal, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2 Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal 100644, CEP 24001-970, Niter´oi, RJ, Brazil (∗ Author for correspondence: e-mail:
[email protected], fax: +5521-2629-2292) Received 19 December 2004; accepted 27 June 2005
Key words: amino acids, marine macroalgae, marine microalgae, nitrogen, protein determination, seaweeds Abstract Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical methods employed. The different extraction procedures (exposure to water, grinding, etc.), protein precipitation using different amounts of 25% trichloroacetic acid and quantification of protein by two different methods and using two protein standards were evaluated. All procedures were tested using freeze-dried samples of three macroalgae: Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocol for protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure water for 12 h, followed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should be done with 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in this study was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima, Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae (Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Comparison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry’s method should be used instead of Bradford’s using bovine serum albumin (BSA) as protein standard instead of casein. This may be related to the reactivity of the protein standards and the greater similarity in the amino acid composition of BSA and algae. The current results should contribute to more accurate protein determinations in marine algae. Introduction Determination of protein content of algae can provide important information on the chemical characteristics of algal biomass. The methods most commonly used to quantify protein are: (i) the alkaline copper method (Lowry et al., 1951); (ii) the Coomassie Brilliant Blue dye method (Bradford, 1976); or (iii) determination of crude protein (N × 6.25). The calculation of protein content by N × 6.25 requires some caution, not always considered by authors using this method. Plant materials, fungi and algae commonly have high concentrations of non-protein nitrogenaceous substances such as pigments (chlorophyll and phycoerythrin), nucleic acids, free amino acids
and inorganic nitrogen (nitrate, nitrite and ammonia) (Louren¸co et al., 1998; Conklin-Brittain et al., 1999; Fujihara et al., 2001) whose presence makes the factor 6.25 unsuitable since it overestimates the actual protein content (Ezeagu et al., 2002). Specific nitrogento-protein conversion factors were recently proposed for 12 marine microalgae (Louren¸co et al., 2004) and 19 seaweeds (Louren¸co et al., 2002), varying from 3.75 for Cryptonemia seminervis a red alga, to 5.72 for Padina gymnospora a brown alga. The determination of protein by the Lowry and Bradford methods is carried out by spectrophotometry. The Lowry method detects protein by a reaction catalyzed by copper, a component of the Folin phenol reactions. The chemical reaction detects peptide
448 bonds and is also sensitive to some amino acids such as tyrosine and tryptophan (Legler et al., 1985). In the Bradford method, the Coomassie Brilliant Blue dye is bound to protein mainly by arginine residues and to a lower degree by histidine, lysine, tyrosine, tryptophan and phenylalanine residues. The binding between the dye and amino acids is attributed to van der Waals forces and hydrophobic interactions (Compton & Jones, 1985). As a consequence, the reactivity of both methods in comparison to a specific protein is strongly influenced by its amino acid composition, since not all amino acids can oxidate equally the Folin phenol reactive or bind to the Coomassie dye (Stoscheck, 1990). The differences in the principles of the methods contribute to making comparison of results available in the literature even more difficult, since the choice of the method to be used is an arbitrary decision. Bovine serum albumin (BSA) is the most used protein standard for calibration curves in spectrophotometry, but many other proteins can be used. Several studies suggest that the Lowry and Bradford analyses produce different measurements of protein when using BSA as the protein standard for samples such as the gut fluid of fish (Crossman et al., 2000), marine invertebrates (Zamer et al., 1989), higher plants (Eze & Dumbroff, 1982) and marine phytoplankton (Clayton et al., 1988). To obtain a more reliable measurement of protein, it would be useful to identify the predominant proteins in the cells (Berges et al., 1993). However, this recommendation has no practical value, considering the difficulty of extracting, purifying and characterising the main proteins present in the cells and subsequently using them as protein standards. Nguyen & Harvey (1994) suggested the use of ribulose-1,5-diphosphate carboxylase (RuDPCase) for calibration curves to analyse samples of photosynthetic organisms, since RuDPCase corresponds to about 15% of the total protein in chloroplasts. Several substances may interfere with both the Lowry and Bradford method, such as phenol and phenolases (Mattoo et al., 1987), glucosamine and detergents (Peterson, 1979) and flavonoids (Compton & Jones, 1985) among many others (see the comprehensive studies of Peterson, 1979; Stoscheck, 1990 on interfering substances). These substances could affect analyses by either increasing the absorbance (overestimating values), or decreasing the measurements by inhibiting the action of specific reagents. However, their influence may be avoided by precipitation of the protein sample with trichloroacetic acid (TCA). Concen-
trations of TCA between 0.18 and 0.34 M can be used to seperate protein from the other extract components, because only protein is precipitated (Clayton et al., 1988). The physical separation among protein, small peptides and free amino acids is especially important, since the analytical methods are sensitive to the last two classes of substances. Thus, protein precipitation with TCA is strongly recommended to avoid the quantification of small peptides and free amino acids (Nguyen & Harvey, 1994) as well as interference by other substances. As many variables are simultaneously involved with protein analysis, the influence of specific factors may be neglected by authors, affecting the accuracy of protein analysis. However, studies focussing on protein analysis in algae are relatively uncommon and experimental data are needed to fill this gap. It is also very important to develop a simple and inexpensive protocol, using low-cost equipment and consumables, in order to make it accessible to everyone interested in data on algal protein: researchers, algae producers, people interested on the nutritional value of algae and so on. In this study, different procedures for extraction and quantification of the protein content of marine algae were evaluated. The specific aims of this study were: (i) to create a protocol for the extraction and quantification of protein of marine algae; (ii) to compare the use of two methods (Lowry and Bradford) for the determination of protein in marine algae; (iii) to evaluate the effects of the time of extraction, the use of grinding and the precipitation of samples with TCA on the quantity of protein extracted; and (iv) to compare the amino acid profile of the algal samples and the protein standards (BSA and casein) used.
Materials and methods Fifteen species of marine algae covering a wide taxonomical range were analysed. The field-collected marine were identified following the checklist of Wynne (1998). Marine microalgae were cultured in the laboratory. The classification below is based on Lee (1999): Chlorophyta 1. Chlorophyceae: Dunaliella tertiolecta Butcher (Volvocales). 2. Ulvophyceae: Caulerpa fastigiata Montagne (Bryopsidales), Codium decorticatum (Woodw.) M. Howe; (Bryopsidales) and Ulva fasciata Delile (Ulvales).
449 Cryptophyta Cryptophyceae: Hillea sp. Schiller (Cryptomonadales). Dinophyta Dinophyceae: Amphidinium carterae Hulburt (Gymnodiniales). Heterokontophyta Bacillariophyceae: Skeletonema costatum (Greville) Cleve (Biddulphiales). Phaeophyceae: Chnoospora minima (K. Hering) Papenfuss (Scytosiphonales), Dictyota menstrualis (Hoyt) Schnetter, H¨ornig et Weber-Peukert (Dictyotales), Padina gymnospora (K¨utzing) Sonder (Dictyotales) and Sargassum vulgare C. Agardh (Fucales). Prymnesiophyta Prymnesiophyceae: Isochrysis galbana Parke (Pavlovales). Rhodophyta Bangiophycidae: Porphyra acanthophora var. acanthophora E. C. Oliveira and Coll (Bangiales). Florideophycidae: Aglaothamnion uruguayense (Taylor) Aponte, Ballantine et Norris (Ceramiales) and Pterocladiella capillacea (S. G. Gmel.) Santelices et Hommersand (Gelidiales). All species of marine macroalgae were collected in June 1998 at Rasa Beach (located in Arma¸ca˜ o de B´uzios, 22◦ 44 S and 41◦ 57 W) and Per´o Beach (located in Cabo Frio, 22◦ 51 S and 41◦ 58 W), Northern Rio de Janeiro State, Brazil. Whole thalli of adult plants were collected early in the morning and washed in the field with seawater to remove epiphytes, sediment and organic matter. Algae were packed in plastic bags and kept on ice until returned to the laboratory. In the laboratory, samples were gently brushed under running seawater, rinsed with distilled water, dried with paper tissue, frozen at −20 ◦ C and freeze-dried. The dried material was powdered manually with the use of mortar and pestle and kept in desiccators containing silicagel and protected from light at room temperature until chemical analysis. Culture of microalgae All microalgal strains used in this study are available at the Elizabeth Aidar Microalgae Culture Collection, Department of Marine Biology, Federal Fluminense University, Brazil. Starter cultures of 50–100 mL in mid-exponential growth phase were inoculated into 2.0 L of seawater, previously autoclaved at 121 ◦ C for 30 min in 3.0 L borosilicate flasks, and enriched with
Conway nutrient solution (Walne, 1966). Each experiment was carried out in four culture flasks, exposed to 300 µmol photons m−2 s−1 (measured with a Biospherical Instruments quanta meter QLS100) from beneath, provided by fluorescent lamps (Sylvania daylight tubes), under a 12:12 h light:dark cycle. Mean temperatures were 23 ± 1 ◦ C in the light period and 20 ± 1 ◦ C in the dark period. Salinity of the culture medium was 32.0‰. Growth rates were calculated daily by direct microscopic cell counting with Fuchs–Rosenthal or Malassez chambers. Cultures were bubbled with filtered air at a rate of 2 L min−1 . The culture medium was not buffered and pH was determined daily. Each culture was sampled in the stationary growth phase only. Cultures were concentrated by centrifugation at 7000 g for 10 min at 15 ◦ C, at least once. Before the last centrifugation, cells were washed with artificial seawater (Kester et al., 1967) prepared without nitrogen, phosphorus and vitamins and adjusted to 15‰ salinity to remove any residual nitrogen from the culture medium. All supernatants obtained for each sample were combined and the cell number was determined in this pool to quantify possible cell losses. The pellets were frozen at −20 ◦ C, freeze-dried (as described above), weighed and stored in desiccators under vacuum and protected from light at room temperature until analysis was done.
Amino acid analysis Samples containing 5.0 mg of protein were acid hydrolysed with 1.0 mL of 6 N HCl in vacuum-sealed hydrolysis vials at 110 ◦ C for 22 h. Norleucine was added to the HCl as an internal standard. Although tryptophan was completely lost with acid hydrolysis and methionine and cystine + cysteine could be destroyed to varying degrees by this procedure, the hydrolysates were suitable for analysis of all other amino acids. The tubes were cooled after hydrolysis, opened, and placed in a descicator containing NaOH pellets under vacuum until dry (5–6 days). The residue was then dissolved in a suitable volume of a sample diR lution Na–S buffer (Beckman Instr.), pH 2.2, filtered through a Millipore membrane (0.22 µm pore size) and analysed for amino acids by ion-exchange chromatography in a Beckman, model 7300 instrument equipped with an automatic integrator. Ammonia content is also presented as it comes from the degradation of some amino acids (e.g. glutamine, asparagine) during acid hydrolysis (Moss´e, 1990).
450 Total nitrogen Total nitrogen (TN) content was determined by CHN analysis. 0.8–1.5 mg freeze-dried samples were combusted in a CHN analyser (Perkin–Elmer, model 2400). Helium was used as carrier gas. Acetanilide (C = 71.09%; N = 10.36%; H = 6.71%) and/or benzoic acid (C = 68.84%; H = 4.95%) were used to calibrate the instrument. Extraction of protein Eight procedures for protein extraction were tested in this study. All procedures start with 50 mg of freezedried algal sample, ground manually with pestle and mortar. Two different volumes of water were tested (1.0 and 4.0 mL), as well as two different incubation periods of samples with water (6 and 12 h). In all the cases samples were kept at 4 ◦ C during the incubation period. In four out of the eight extraction procedures, samples were also ground using a Potter homogeniser (Marconi, model MA099) after the incubation with water. Samples were water-ground with a glass pestle and a teflon mortar at medium speed. During grinding, samples were kept cool by the use of a circulating cooling bath through the pestle. The grinding of samples was started 1 h before the end of incubation of the samples with water. Six replicates were prepared for each treatment and each species. Procedures for protein extraction are based on Fleurence et al. (1995) with some modifications (e.g. speed of centrifugation, grinding of the samples, time of incubation). The specific procedures for protein extraction analysed in this study are as follows: Procedure I. Algal samples were immersed in 1 mL of ultra-pure water for 12 h. After the incubation period, suspensions were centrifuged at 4 ◦ C, 15,000 g for 20 min. Supernatants were collected for protein assay and the pellets re-extracted with 1.0 mL 0.1 N NaOH with 0.5% β-mercaptoethanol (v/v). The mixture of NaOH and pellets were kept at room temperature for 1 h with occasional manual shaking and then centrifuged at 21 ◦ C, 15,000 g for 20 min. The second supernatants were combined with the first ones and the pellets were discarded. The final volume of the extract was 2.0 mL. Procedure II. Similar to procedure I, with one additional step included: the grinding of samples with a Potter tissue homogeniser for 5 min, 1 h before the
end of the incubation period. Seven millilitre of ultrapure water was added to the system to rinse the Potter homogeniser after grinding each sample to recover all water-ground material. After this step, samples were treated as described after the end of the incubation period for procedure I. The final volume of the extract was 9.0 mL. Procedure III. This treatment is similar to procedure I, differing by the use of 4 mL of water to incubate dried samples, instead of 1 mL. The final volume of the extract was 5.0 mL. Procedure IV. This treatment is similar to procedure II, differing by the use of 4 mL of water for incubating dried samples, instead of 1 mL. Four millilitre of ultra-pure water were added to the system to rinse the Potter homogeniser after grinding each sample to recover all water-ground material. After this step, samples were centrifuged as described for procedure I. The final volume of the extract was 9.0 mL. Procedure V. Similar to procedure I, differing only due the incubation period: 6 h instead of 12 h. The final volume of the extract was 2.0 mL. Procedure VI. Dried samples are treated such as in procedure II, except by the shorter incubation period: 6 h instead of 12 h. The final volume of the extract was 9.0 mL. Procedure VII. Similar to procedure III, differing only due to the shorter incubation period: 6 h instead of 12 h. The final volume of the extract was 5.0 mL. Procedure VIII. Samples were treated as described in procedure IV, differing only by the shorter incubation period: 6 h instead of 12 h. The final volume of the extract was 9.0 mL. A summary of the procedures to extract algal protein is shown in Table 1. Table 1. Summary of the procedures for extracting protein used in this study Treatment
Volume of water (ml)
Period of incubation (h)
Grinding
Procedure I Procedure II Procedure III Procedure IV Procedure V Procedure VI Procedure VII Procedure VIII
1.0 1.0 4.0 4.0 1.0 1.0 4.0 4.0
12 12 12 12 6 6 6 6
No Yes No Yes No Yes No Yes
451 The use of 1.0 mL 0.1 N NaOH with 0.5% βmercaptoethanol (v/v) for re-extracting the pellets was adopted in all the eight procedures tested, independent of the time of incubation and volume of ultra-pure water used to incubate the samples. Precipitation of protein Protein precipitation was followed Berges et al. (1993). Two proportions of cold 25% trichloroacetic acid (TCA) (4 ◦ C) added to the extracts were tested: 2.5:1 and 3.0:1 (TCA:homogenate, v/v). Tubes containing TCA and homogenate were kept in an ice bath for 30 min and then centrifuged for 20 min at 4 ◦ C (15,000 g). Supernatants were discarded, pellets were washed with cold 10% TCA (4 ◦ C) and centrifuged again. Pellets formed after the second centrifugation were suspended in 5% TCA at room temperature, in a proportion of 5:1 (5% TCA:precipitate, v/v) and centrifuged at 21 ◦ C (15,000 g) for 20 min. Supernatants were discarded and pellets were kept in the tubes until quantification of protein was done a few minutes later. When the protein analysis was not performed immediately, pellets were stored at −20 ◦ C until further analysis, following Dortch et al. (1984). Precipitated protein was suspended in 0.5 mL 1.0 N NaOH and 2.0 mL 0.1 N NaOH for the a Bradford and Lowry assays, respectively. Aliquots were also collected from the crude extracts obtained before the precipitation with TCA to perform protein analysis by the Lowry method without previous precipitation. Protein analysis In the Lowry method, the Folin–Ciocalteu reactive (Folin & Ciocalteu, 1927) (Sigma Co.) was diluted in two volumes of ultra-pure water (1:2) and 0.5 mL of the diluted reactive was added to 1.0 mL of sample, previously mixed with 5.0 mL of the reactive “C” [50 volumes of reactive “A” (2.0% Na2 CO3 + 0.1 N NaOH) + 1 volume of reactive “B” (1/2 volume of 0.5% CuSO4 5H2 O + 1/2 volume of 1.0% C4 H4 NaO6 4H2 O)]. After the addition of each reactive, samples were stirred for 2 s in a test tube stirrer. Absorbance was measured at 750 nm, 35 min after the start of the chemical reaction at room temperature. In the Bradford assay, the Coomassie Brilliant Blue dye G-250 (CBBG) binds to the protein. The binding of the dye with the protein is very quick and the protein-dye complex remains soluble for 1 h. One hundred milligram of CBBG (Sigma Co.) was dissolved in
50 mL 95% ethanol (Merck Co.) with a further addition of 100 mL 85% H3 PO4 (Merck Co.). The solution was diluted with ultra-pure water to 1.0 L. Five millilitre of the reactive was used for each 0.1 mL sample. Absorbance was measured at 595 nm, 5 min after the start of the chemical reaction at room temperature. Calibration curves were prepared using bovine serum albumin (BSA) (Sigma Co.) and casein (Sigma Co.) at maximum concentrations of 100 µg mL−1 (Lowry method) and 100 µg 0.1 mL−1 (Bradford method). Casein was diluted in ultra-pure water plus some drops of 0.1 N NaOH. All measurements were done using a Shimadzu, model UV Mini 1240 spectrophotometer. In addition to colorimetric assays of protein, crude protein for each species was also calculated using specific nitrogen-to-protein conversion factors proposed by Louren¸co et al. (2002, 2004) as follows: A. carterae (5.13), A. uruguayense (3.94), C. decorticatum (5.34), C. fastigiata (4.52), C. minima (5.70), D. menstrualis (4.55), D. tertiolecta (4.39), Hillea sp. (4.93), I. galbana (5.07), P. acanthophora var. acantophora (4.47), P. capillacea (4.78), P. gymnospora (5.72), S. costatum (4.53), S. vulgare (5.53), and U. fasciata (5.59). Statistical analysis The results were analysed by one-way analysis of variance (ANOVA) with significance level α = 0.05 (Zar, 1996) followed, where applicable, with Tukey’s multiple comparison test. In some cases, Student’s t-test was used instead of ANOVA when comparing only two treatments for each variable.
Results Tests of protein extraction The use of the Potter homogeniser produced remarkable differences in the extraction of protein for the three species tested. In all cases, significantly ( p < 0.001) higher concentrations of protein was obtained in the treatment with the use of the Potter homogeniser (Figures 1A–C). For Sargassum vulgare (Figure 1B) differences in values obtained for samples extracted with and without the Potter homogeniser were about 50%. Higher values of protein were also obtained for samples of Porphyra acanthophora var. acanthophora and Ulva fasciata when extracted with the Potter homogeniser.
452 The precipitation of protein No differences in the precipitation of protein were observed for the two TCA:homogenate ratios using the Lowry (0.12 ≤ p ≤ 0.70) and Bradford (0.14 ≤ p ≤ 0.97) methods. The quantification of protein in the tests
Figure 1. Quantification of protein in three species of marine macroalgae (A) Porphyra acanthophora var. acanthophora, (B) Sargassum vulgare and (C) Ulva fasciata by the Lowry (Lwy) and Bradford (Bdf) methods, using bovine serum albumin (BSA) and casein (CAS) as protein standards in the calibration curves. Three variables evaluated: (i) the water volume used to incubate samples (1 and 4 mL); (ii) the length of the incubation period for the extraction of protein (6 and 12 h); and (iii) the use of a Potter homogenizer for grinding samples. All assays included the precipitation of protein with 25% TCA, in a proportion of 2.5:1 (TCA:homogenate). Mean ± S.D. (n = 6).
Higher concentrations of protein were obtained when 4.0 mL of water was used for U. fasciata ( p < 0.001) (Figure 1C), and a longer period of incubation (12 h) for S. vulgare ( p = 0.002) (Figure 1B). There were no differences for P. acanthophora var. acanthophora (Figure 1A) incubated in wither 1.0 or 4.0 mL for 6 h ( p = 0.52).
The results show large differences for all the three species between the two protein quantification methods; values obtained with the Lowry method were always higher in all comparisons evaluated ( p < 0.01) (Figures 1A–C). The use of BSA in calibration curves seems to generate higher values in the samples of P. acanthophora var. acanthophora ( p < 0.02). However for U. fasciata, using the Lowry method ( p = 0.56) and S. vulgare with the Bradford method ( p = 0.06), no differences were found when comparing results with BSA or casein as calibration standards. In addition to the general analyses described above, some tests on samples spiked with protein were performed to assess any possible effects of endogenous algal proteases in the samples which could partially destroy protein during incubation (P´erez-Llor´enz et al., 2003). Tests were carried out for all the three algae at same dilutions used to extract and quantify algal protein. Controlled amounts of BSA were used as an internal standard: 2.5 mg of BSA were added to each flask in which the algal material was incubated, such as in procedure IV. Controls included incubation of BSA with no algae, following the same steps described for algal samples and direct dilution and measurement of BSA, without incubation. The results (data not presented) showed no loss of protein after the incubation period in all experiments carried out ( p ≥ 0.083), indicating no activity of algal proteases at 4 ◦ C, the incubation temperature used. Protocol for extraction and precipitation of protein Results suggest the use of a general protocol involving extraction of protein from algal samples using 4.0 mL of ultra-pure water, for 12 h and grinding of samples with a Potter homogeniser. Samples should be precipitated with TCA:homogenate (2.5:1 v/v). A diagram of the protocol is shown in Figure 2. This protocol was used for protein determination of the other algal species using both the Bradford and Lowry methods, using
453
Figure 2. Diagrammatic representation of the protocol for extraction and precipitation of algal protein developed in this study.
454 BSA and casein as protein standards in the calibration curves. Amino acid profile of the algae and protein standards The comparison of the amino acid profile of the algal species (Table 2) shows great differences for aspartic acid, glutamic acid and arginine. On the other hand, some amino acids, such as glycine and leucine, had similar values for the 15 species studied. The comparison of the algal amino acid profile with the protein standards show that the concentration of glutamic acid in casein, and lysine in BSA, are higher than those reported for all the algae. Both protein standards were lower in methionine compared to the algae. Casein and BSA also show remarkable differences with each other regarding some amino acids, such as alanine (higher concentration in BSA) and proline (higher concentration in casein).
fastigiata, casein as protein standard). For all species, values obtained with BSA as the protein standard were higher than those obtained with casein (Table 3). Crude extracts analysed using the Lowry method always gave higher values than those obtained with precipitated samples ( p < 0.01), except for A. uruguayense and Hillea sp. (Table 3). The 15 algae species showed great differences regarding total nitrogen and crude protein (Table 3). Microalgae had smaller variations in the total N, varying from 3.35% (I. galbana) to 4.69% (A. carterae). Variations in the total N were greater in the macroalgae, ranging from 1.94% (C. minima) to 5.68% (A. uruguayense). The calculations of crude protein gave wide variations among species, varying from 10.52% in C. decorticatum to 25.04% in Hillea sp. (Table 3). The sum of amino acid residues varied from 9.99% (C. minima) to 20.11% (Hillea sp.). Microalgae tended to have higher percentages of amino acid residues than macroalgae.
Quantification of protein Discussion The highest percent of protein was measured in the red algae A. uruguayense (15.6 ± 0.3% of the dry matter), followed by the cryptomonad Hillea sp. (15.3 ± 0.6%) (Table 3). The other microalgae had similar protein contents, varying from 11.4 ± 1.0%. (D. tertiolecta) to 10.1 ± 0.8% (I. galbana). The edible red algae P. acanthophora var. acanthophora had 8.9 ± 0.7% of protein and P. capillacea had the lowest protein content among the red algae (4.2 ± 0.3%). In the brown algae, small variations in protein content were found (8.7, 7.8 and 6.9% for P. gymnospora, C. minima and S. vulgare, respectively), except for D. menstrualis, which had a lower protein content (4.0 ± 0.2%). The green macroalgae had protein contents varying from 5.5% (C. fastigiata) to 7.3% (U. fasciata) (Table 3). Confirming the same trend obtained with the initial results for the three macroalgae (P. acanthophora var. acanthophora, S. vulgare and U. fasciata), all other species gave significantly higher values of protein when quantified using the Lowry method compared to the Bradford method. In some cases (e.g. C. fastigiata), differences between mean values were higher than 50% (Table 3). For the microalgae, the variation between the two methods was less, varying from 1.5 (I. galbana, BSA as protein standard) to 1.9 (A. carterae, BSA and casein as protein standard). For the macroalgae, the Lowry:Bradford ratio varied from 1.5 (D. menstrualis, BSA and casein as protein standard) to 3.2 (C.
Extraction and precipitation of algal protein Protein content of the three main algae species tested in this study varied greatly depending on the different extraction procedures tested. The efficiency of extraction seems to be influenced directly by two main factors: the chemical composition of the species and its morphological and structural characteristics. The chemical composition of the three species is very distinct (Louren¸co et al., 2002) and, in theory, this may lead to differences in the protein content. S. vulgare is a branched algae and possesses a hard and leathery thallus, while P. acanthophora var. acanthophora and U. fasciata have flattened soft thalli. In the present study, the effects of lyophylisation on the thalli should be also considered since freeze-dried samples tend to be more difficult to extract, especially leathery species such as C. minima and S. vulgare. This means that better preservation by freeze-drying makes them more difficult for further protein extraction. This problem can be solved by grinding samples with Potter homogeniser. For P. acanthophora var. acanthophora lyophilisation, the main factor influencing the yield in the protein extraction was the use of grinding. This is probably related to the thallus form of this species. Ulva fasciata has the same kind of thallus, but the use of
455 Table 2. Amino acid profile of 15 species of marine algae and two standards of pure protein: bovine serum albumin (BSA) and caseina Protein standards Amino acid
BSA
Casein
Chlorophyta C. fastigiata
Cryptophyta
C. decorticatum D. tertiolecta U. fasciata Hillea sp.
Cisteic acid
4.5 ± 0.4
0.9 ± 0.5
2.8 ± 0.3
1.1 ± 0.3
0.7 ± 0.2
Aspartic acid
9.6 ± 0.2
6.4 ± 0.3
8.8 ± 0.8
10.7 ± 0.1
12.3 ± 0.5
Treonine
4.8 ± 0.2
3.7 ± 0.2
4.7 ± 0.5
6.0 ± 0.1
4.6 ± 0.2
5.2 ± 0.2
4.6± 0.0
Serine
3.8 ± 0.4
4.8 ± 0.3
6.0 ± 0.6
5.0 ± 0.2
3.6 ± 0.1
5.9 ± 0.7
3.8 ± 0.0
5.5 ± 0.3
Glutamic acid 16.7 ± 0.3 21.1 ± 0.8
10.4 ± 1.1
12.0 ± 0.9
12.8 ± 0.4
12.9 ±0.4 12.1 ± 0.0
13.6 ± 0.3
Proline
4.7 ± 0.3 11.3 ± 0.6
5.1 ± 0.4
4.8 ± 0.7
4.9 ± 0.2
4.7 ± 0.1
3.5± 0.0
4.2 ± 0.3
Glycine
1.7 ± 0.3
1.6 ± 0.2
6.9 ± 0.8
7.2 ± 0.5
5.8 ± 0.1
6.7 ± 0.2
7.2± 0.5
5.1 ± 0.2
Alanine
5.4 ± 0.2
2.6 ± 0.3
6.0 ± 0.6
8.8 ± 0.6
7.1 ± 0.2
8.7 ± 1.1
6.9± 0.0
7.3 ± 0.2
Valine
5.7 ± 0.1
6.0 ± 0.2
6.0 ± 0.5
6.2 ± 0.0
5.7 ± 0.8
5.9 ± 0.6
5.9 ± 0.0
6.2 ± 0.1
Methionine
N.D.
0.5 ± 0.0
1.0 ± 0.2
0.7 ± 0.5
2.8 ± 0.3
0.9 ± 0.1
2.8 ±0.0
1.9 ± 0.2
Isoleucine
2.4 ± 0.1
4.7 ± 0.2
3.9 ± 0.4
3.8 ± 0.6
4.3 ± 0.1
4.0 ± 0.7
4.9± 0.1
4.0 ± 0.1
Leucine
10.9 ± 0.1
8.8 ± 0.3
8.5 ± 0.9
8.4 ± 1.1
8.3 ± 0.1
7.9 ± 0.7
7.9± 0.3
8.4 ± 0.1
Tyrosine
3.8 ± 0.1
4.1 ± 0.1
3.8 ± 0.4
2.1 ± 0.3
3.2 ± 0.1
3.3 ± 0.7
5.5± 0.2
3.8 ± 0.3
Phenylalanine
5.8 ± 0.1
4.8 ± 0.2
6.4 ± 0.6
5.0 ± 0.7
5.6 ± 0.2
5.3 ± 0.1
5.6 ± 0.4
5.4 ± 0.1
Histidine
3.5 ± 0.6
4.0 ± 0.8
2.2 ± 0.7
3.3 ± 0.2
2.1 ± 1.2
2.5 ± 0.5
1.9± 0.1
3.0 ± 0.4
11.4 ± 0.1
7.4 ± 0.2
6.9 ± 0.6
6.3 ± 0.1
5.5 ± 0.3
5.2 ± 0.4
5.3± 0.0
7.1 ± 0.3
Arginine
5.1 ± 0.5
3.7 ± 0.7
6.4 ± 0.9
5.0 ± 0.4
5.6 ± 0.6
5.7 ± 0.9
4.0± 0.3
6.5 ± 0.2
Ammonia
1.0 ± 0.2
1.5 ± 0.2
1.0 ± 0.2
1.6 ± 0.1
2.5 ± 0.1
1.8 ± 0.1
1.8± 0.0
0.6 ± 0.0
Total
99.8 ± 3.0 96.5 ± 6.7
94.8 ± 5.5
96.4 ± 4.7
94.9 ± 5.5
98.7 ± 2.3 95.2 ± 2.5
98.8 ± 3.6
Amino acid
C. minima D. menstrualis P. gymnospora S. costatum
S. vulgare
I. galbana A. uruguayense P. acanthophora P. capillacea
Lisine
Heterokontophyta
0.5 ± 0.1
0.8 ± 0.2
0.2 ± 0.0
13.4 ± 1.1 13.1 ± 0.2
9.1 ± 0.2
Prymnesiophyta
0.5 ± 0.0
0.9 ± 0.3
0.3 ± 0.0
0.6 ± 0.2
Aspartic acid 12.0 ± 0.8 14.5 ± 0.7
12.8 ± 2.5
13.4 ± 0.1
10.6 ± 1.3
Cisteic acid
0.5 ± 0.1
Dinophyta A. carterae
0.6 ± 0.0
5.1 ± 0.1
Rhodophyta
0.9 ± 0.3
1.2 ± 0.3
0.7 ± 0.1
12.6 ±0.7 13.2 ± 1.8
12.5 ± 2.1
11.6 ± 2.8 5.2 ± 0.9
Treonine
5.1 ± 0.3
5.0 ± 0.0
5.1 ± 0.6
5.2 ± 0.1
4.4 ± 0.7
5.1 ± 0.4
5.4± 0.6
5.8 ± 0.3
Serine
6.0 ± 0.5
6.8 ± 0.4
5.0 ± 0.6
4.7 ± 0.1
4.7 ± 0.7
4.1 ± 0.4
5.2 ± 0.2
5.3 ± 0.4
5.7 ± 1.4
Glutamic acid 14.8 ± 1.4 12.6 ± 0.4
13.1 ± 1.4
13.5 ± 0.0
17.4 ± 0.4
12.1 ±0.2 14.9 ± 1.5
12.9 ± 4.3
14.7 ± 0.2
Proline
4.3 ± 0.4
4.8 ± 0.1
4.3 ± 0.7
3.7 ± 0.0
4.2 ± 0.7
4.1 ± 0.5
4.9± 0.4
4.6 ± 0.1
4.9 ± 0.6
Glycine
6.0 ± 0.4
6.0 ± 0.0
6.0 ± 0.9
6.2 ± 0.1
5.3 ± 0.9
5.8 ± 0.2
6.5± 0.1
7.1 ± 1.4
6.0 ± 0.9
Alanine
7.9 ± 0.8
6.6 ± 0.2
6.9 ± 0.5
6.7 ± 0.1
6.8 ± 1.1
7.4 ± 0.3
7.5± 0.7
8.8 ± 1.2
7.2 ± 1.4
Valine
5.7 ± 0.4
5.2 ± 0.1
5.3 ± 0.6
5.9 ± 0.0
5.4 ± 0.9
6.4 ± 0.2
6.0 ± 0.7
6.4 ± 0.2
5.5 ± 1.8
Methionine
2.0 ± 0.3
1.3 ± 0.2
1.0 ± 0.4
2.6 ± 0.1
1.7 ± 0.3
2.6 ± 0.1
0.7± 0.3
1.1 ± 0.1
1.1 ± 0.1
Isoleucine
3.9 ± 0.4
4.3 ± 0.0
4.3 ± 0.3
5.7 ± 0.0
4.3 ± 0.8
5.1 ± 0.2
4.7± 0.2
4.1 ± 0.8
3.7 ± 0.6
Leucine
7.9 ± 0.6
8.6 ± 0.1
8.5 ± 1.1
8.3 ± 0.1
8.2 ± 1.4
9.3 ± 0.3
8.2± 1.2
8.1 ± 0.9
6.8 ± 1.3
Tyrosine
1.8 ± 0.3
2.6 ± 0.1
2.1 ± 0.4
3.2 ± 0.1
1.8 ± 0.2
3.4 ± 0.2
2.4± 0.3
2.4 ± 0.4
3.7 ± 0.4
Phenylalanine
4.9 ± 0.3
5.5 ± 0.1
5.2 ± 0.7
6.1 ± 0.1
4.9 ± 0.8
5.9 ± 0.1
5.2 ± 0.6
4.7 ± 1.0
5.3 ± 0.9
Histidine
2.0 ± 0.2
2.2 ± 0.1
2.1 ± 0.5
1.6 ± 0.1
1.6 ± 0.3
2.0 ± 0.2
2.4± 0.5
3.0 ± 0.6
3.5 ± 0.5
Lisine
5.0 ± 0.4
4.6 ± 0.2
5.4 ± 0.8
4.6 ± 1.2
5.0 ± 0.9
5.4 ± 0.4
6.2 ± 0.9
6.3 ± 1.4
7.9 ± 0.8
Arginine
4.2 ± 0.3
5.1 ± 0.2
5.0 ± 0.6
4.1 ± 0.0
3.9 ± 0.6
5.8 ± 0.9
4.7± 0.2
4.8 ± 0.1
5.6 ± 0.6
Ammonia
1.4 ± 0.2
1.2 ± 0.1
1.5 ± 0.2
2.4 ± 0.0
1.3 ± 0.1
1.9 ± 0.1
1.8± 0.3
1.9 ± 0.1
95.2 ± 4.7 98.0 ± 2.6
94.5 ± 6.4
95.8 ± 2.3
94.0 ± 4.6
98.3 ± 3.2 96.6 ± 4.6
99.2 ± 6.1
Total a Results
1.7 ± 0.56 99.0 ± 5.3
are expressed as percentage of amino acid per 100 g of algal protein (or pure protein for the two standards) and represent the real recovery of amino acids after analysis. Concentrations of ammonia correspond to nitrogen recovery from some amino acids destroyed during acid hydrolysis. Values indicate the mean of three replicates ± S.D. (n(3). N.D.: not detected.
456 Table 3. Total nitrogen, total amino acid residues and protein content of marine algae, as percentage of the dry mattera Lowry precipitation with TCA 2.5:1
Bradford precipitation with TCA 2.5:1
Lowry row extract, no precipitation
Species
Total nitrogen
BSA
Casein
BSA
Casein
BSA
Casein
Total amino Crude acid residues protein
Chlorophyta C. fastigiata C. decorticatum D. tertiolecta U. fasciata
4.32 ± 0.36 2.13 ± 0.10 4.18 ± 0.09 2.29 ± 0.02
5.47 ± 0.34 7.12 ± 0.53 11.4 ± 0.99 7.30 ± 0.84
5.29 ± 0.33 6.88 ± 0.51 11.0 ± 0.95 7.05 ± 0.81
1.74 ± 0.08 4.49 ± 0.37 6.86 ± 0.26 2.60 ± 0.19
1.63 ± 0.07 4.24 ±0.35 6.48 ±0.25 2.43 ± 0.23
7.52 ± 0.58 7.55 ± 0.08 11.0 ± 0.13 7.55 ± 0.10
7.27 ± 0.56 7.30 ± 0.08 10.6 ± 0.12 7.37 ± 0.09
13.50 ± 2.31 10.93 ± 1.06 17.14 ± 0.70 11.03 ± 0.98
19.53 11.37 18.35 12.80
Cryptophyta Hillea sp.
5.08 ± 0.26 15.3 ± 0.60 14.8 ± 0.58 8.54 ± 0.35 8.07 ± 0.33 13.1 ± 0.51 12.7 ± 0.49 20.11 ± 2.22 25.04
Dinophyta A. carterae
4.69 ± 0.05 10.2 ± 0.09 9.85 ± 0.09 5.49 ± 0.26 5.18 ± 0.25 10.8 ± 0.12 10.5 ± 0.12 15.84 ± 1.72 24.06
Heterokontophyta C. minima D. menstrualis P. gymnospora S. costatum S. vulgare
1.94 ± 0.10 3.26 ± 0.15 2.41 ± 0.14 3.41 ± 0.22 2.08 ± 0.14
Prymnesiophyta I. galbana
3.35 ± 0.20 10.1 ± 0.85 9.75 ± 0.82 6.58 ± 0.49 6.22 ± 0.46 11.1 ± 0.26 10.7 ± 0.25 15.83 ± 0.09 16.98
7.83 ± 0.33 4.04 ± 0.17 8.69 ± 0.72 11.1 ± 0.68 6.91 ± 0.15
7.57 ± 0.32 3.90 ± 0.16 8.40 ± 0.70 10.7 ± 0.66 6.68 ± 0.14
3.56 ± 0.23 2.72 ± 0.21 4.79 ± 0.45 6.43 ± 0.39 3.19 ± 0.19
3.36 ± 0.22 2.56 ±0.20 4.53 ± 0.43 6.07 ± 0.37 3.00 ± 0.18
10.0 ± 0.20 7.01 ± 0.13 11.9 ± 0.50 11.5 ± 0.75 8.77 ± 0.12
9.67 ± 0.20 6.77 ± 0.13 11.5 ± 0.48 11.1 ± 0.72 8.47 ± 0.12
9.99 ± 0.80 10.35 ± 0.31 12.55 ± 1.55 14.30 ± 1.76 11.0 ± 1.54
11.06 14.83 13.78 15.40 11.50
Rhodophyta A. uruguayense 5.68 ± 0.03 15.7 ± 0.33 15.1 ± 0.32 10.2 ± 0.32 9.48 ±0.30 12.2 ± 0.22 11.7 ± 0.21 17.22 ± 1.88 22.38 P. acanthophora 3.68 ± 0.04 4.19 ± 0.29 4.05 ± 0.28 2.60 ± 0.13 2.45 ±0.12 6.26 ± 0.18 6.05 ± 0.17 11.83 ± 1.58 16.45 P. capillacea 3.24 ± 0.10 8.94 ± 0.53 7.98 ± 0.49 4.62 ± 0.18 4.23 ± 0.19 11.7 ± 0.38 10.9 ± 0.35 12.11 ± 3.00 15.49 a Protein
was determined by different methods, with BSA and casein as protein standards. Analysis by Lowry’s method was also made with notprecipitated samples. Data represent the mean of six replicates ± S.D. (n(6), except for total nitrogen and amino acid residues (n(3).
a greater volume of water seems to improve the extraction of protein. Differences in the behaviour of the two flattened algae may be related to the chemical composition and thallus morphology, since U. fasciata has two layers of cells compared to P. acanthophora var. acanthophora with only one layer of cells. For the extraction of protein from the branched and hard thallus of the brown algae S. vulgare, 12 h incubation in water seems to be important. Even using a greater volume of water (4 mL), an incubation period of 6 h was not enough to soften the thalli. Fragments of S. vulgare thallus were ground more easily with the Potter homogeniser after 12 h of exposure to water. As the precipitation of protein with TCA: homogenate (3.0:1 and 2.5:1 v/v) gave no significantly different results and therefore a TCA:homogenate ratio of 2.5:1 (v/v) is recommended in order to save reagent.
Amino acid profile, non-protein nitrogen and protein content of algae In this study we assume that the actual concentrations of protein in the samples are calculated from the sum of amino acid residues (Tables 1 and 2), a widely accepted procedure since the 1970s (Heidelbaugh et al., 1975). After acid hydrolysis, all proteins are destroyed, even those associated with other macromolecules and biological membranes. The values for the total amino acid residues were calculated by summing up the amino acid masses retrieved after acid hydrolysis (total amino acid), less the water mass (18 g in 1 M of each amino acid) incorporated into each amino acid after disruption of the peptide bonds. Total amino acid analysis involves some errors, such as the total (tryptophan) or partial (methionine and cysteine) destruction of some amino acids, as well as the impossibility of identifying the contribution of free amino acids in the samples. However, it indicates the maximum
457 possible concentration of protein in the sample considering that all amino acid are in protein, providing a good reference point for the protein concentrations measured by the Bradford and Lowry assays. In our results, values for amino acid residues were similar to the estimated crude protein, suggesting the suitability of the nitrogen-to-protein conversion factors calculated by Louren¸co et al. (2002, 2004). Exceptions to this are C. fastigiata and A. carterae (Table 3), which showed values for crude protein ca. 30% higher than those for the sum of amino acid residues. This difference probably results from the presence of high concentrations of non-protein nitrogen, presumably transient stocks of inorganic nitrogen (Lav´ın & Louren¸co, unpublished data). The sum of amino acid residues indicates that microalgae, independent of the taxonomic group, tend to accumulate higher concentrations of protein than macroalgae. This fact may be related to the high concentration of nitrogen in the culture medium (as well as other dissolved nutrients), growth conditions in the laboratory and the higher surface area:volume ratios found in microalgae (Hein et al., 1995). Dried samples of microalgae are better exposed to solvents and to grinding during the extraction procedures, while macroalgal samples have to be powdered before the start of the extraction. This factor may produce a more efficient protein extraction. For many macroalgae, the combined concentration of glutamic acid and aspartic acid represents 40% of total amino acids, agreeing with data obtained for the edible red algae Palmaria palmata, in which glu and asp represent 39.6% of total amino acids (GallandIrmouli et al., 1999). For microalgae, the sum of asp and glu represent mean values of about 20% of the total amino acids for Skeletonema costatum, Dunaliella tertiolecta and Thalassiosira pseudonana (Brown, 1991). For Ulva rigida and U. rotundata, percentages of these two amino acid may represent from 26 to 32% of the total amino acids (Fleurence et al., 1995). In the present study, values for asp + glu varied from 19.2% (C. fastigiata) to 28.1% (A. uruguayense) (Table 2). For microalgae, the fraction represented by these two amino acids varied from 23.1% (A. carterae) to 26.9% (S. costatum) of the total amino acids (Table 2). The set composed of the essential amino acids in samples varied from 36.3% (S. vulgare) to 44.0% (C. fastigiata), with a mean value of 40.2% of the total amino acids. Concerning nutritional properties, these species show concentrations of essential amino acids comparable to those commonly described to soybean pro-
tein, which possesses 36.0% of the total amino acid (Galland-Irmouli et al., 1999). High concentrations of non-protein N may result in overestimation of protein (Zamer et al., 1989). According to Louren¸co et al. (2004), concentrations of non-protein N vary widely during growth in cultures of microalgae, commonly fluctuating from 15 to 30% of the total N. In the present study, the occurrence of high concentrations of the total N was not mirrored by high total amino acid concentrations in some species such as A. carterae, A. uruguayense, C. fastigiata, and Hillea sp., which is explained by the presence of large amounts on non-protein N. The determination of crude protein should be based on the use of specific nitrogen-to-protein conversion factors as proposed by Louren¸co et al. (2002, 2004). In addition, results obtained for the total amino acid residues and precipitated and non-precipitated extracts with the Lowry method suggest the influence of variable concentrations of free amino acids and small peptides in the samples. This finding indicates that precipitation of the samples is a fundamental step during protein analysis. Data of protein content in macroalgae from the tropical and subtropical coastal environments frequently show lower concentrations (Kaehler & Kennish, 1996; Wong & Cheung, 2000). In some Brazilian environments Ramos et al. (2000) found that the percentage of protein (N × 6.25) in 14 seaweeds varied from 2.30 to 25.6% of dry weight. Despite the overestimation of protein content caused by the use of the factor 6.25 (Louren¸co et al., 2002), values obtained by Ramos et al. (2000) indicated predominantly low concentrations of protein. This trend may be related to the natural characteristics of Brazilian marine environments; predominantly oligotrophic, with low availability of N (Oliveira et al., 1997; Ovalle et al., 1999). As a consequence, low concentrations of protein would be accumulated by natural populations of macroalgae. In this context, our data of protein concentration in macroalgae are in accordance with the information available in the literature (e.g. Wong & Cheung, 2001; McDermid & Stuercke, 2003). On the other hand, the relative low content of protein in microalgae results from the physiological state of the species. All microalgae were sampled in stationary growth phase when percentages of protein in cells decreased due to depletion of dissolved nutrients in the culture medium (Louren¸co et al., 1998). Low protein levels were found in Pterocladiella capillacea using both the Lowry and Bradford
458 methods. The values obtained are equivalent to 1/3 of those determined from the sum of the amino acid residues, being the lowest protein concentrations among all red macroalgae tested. This seems to indicate inefficient extraction using the procedures developed in this study for this macroalgae. We hypothesise that the extraction of protein in this species might be influenced by the presence of phycocolloids, especially agarans. P. capillacea is a good source of agar (McHugh, 1991) and is probably the most abundant macromolecule in this alga. During the water extraction step, it was possible to extract variable quantities of agar visible, as the occurrence of gels in several steps of protein extraction, mainly when using the Potter homogeniser and after the centrifugation at 4 ◦ C of the ground samples. It is possible that the gels can trap part of the protein extracted, giving low values in the spectrophotometric determination of protein. This kind of analytical problem may be present in other agarophytes as well as carragheenan-producing species. The presence of large amounts of anionic polysaccharide in the cell walls reduces protein solubility during extraction (Fleurence, 1999). Further studies are needed to develop better procedures for protein extraction in phycocolloid-rich species.
with protein occurs mainly with the two amino acids, arginine and phenylalanine, and this fact seems to contribute to lower protein measurements. Our results with Bradford’s method agree with Kaehler and Kennish (1996). These authors found predominantly low values for some seaweeds (from 1.3 to 12.6%) from Hong Kong using the Bradford method. In contrast, the Folin–Ciocalteu reagent used in the Lowry assay interacts with all peptide bonds and also with some amino acids. As a result, the quantification of protein tends to be greater. The differences in amino acid composition among protein standards and algae have important implications regarding protein reactivity and quantification in algal samples. Despite the good linearity obtained with both protein standards (BSA and casein), our data suggest that casein has a slightly smaller reactivity than BSA resulting in a smaller quantification of protein. The two protein standards have extremely different amino acid composition and the reactivity of them in each method tends to be different due the functional groups that they present (Morrison & Boyd, 2003). Functional groups change the charge and the geometry of neighbouring atoms, affecting the reactivity of the whole molecule (Morrison & Boyd, 2003).
Lowry × Bradford methods and the influence of the amino acid profile of samples and standards
Conclusions
Some authors suggest that Bradford’s method would generate lower protein values for a large number of organisms compared to Lowry’s method. Calculating the Lowry:Bradford ratio from data by Eze & Dumbroff (1982) for leaves of bean plants gives a ratio of 1.4. For the diatom T. pseudonana, Clayton et al. (1988) found ratios varying from 1.8 to 2.0, while Berges et al. (1993) determined a ratio of 1.2 for the same microalgae. The present results confirm the general trends found by those authors, but we found higher Lowry:Bradford ratios for most of the species. Our results varied from 1.5 (I. galbana, BSA as the protein standard) to 3.2 (C. fastigiata, casein as the protein standard). The trend of obtaining lower concentrations of protein using Bradford’s method may be related to the binding of the dye Coomassie Brilliant Blue-G250 to both basic and aromatic amino acid residues (Compton & Jones, 1985). Most of the algae show relatively low concentrations of the two amino acids (tyrosine and tryptophan) as well as the two basic amino acids (lysine and histidine). Thus, the binding of the dye
The use of 4.0 mL of water to incubate algal samples for 12 h, combined with grinding of the samples with a Potter homogeniser is strongly recommended. This procedure results in better extraction of protein from algal samples of different species independent of their morphological and biochemical characteristics. As a consequence, this procedure can be applied widely to many algal species. The precipitation of protein should be done with 25% trichloroacetic acid in the ratio of 2.5:1 (TCA:homogenate). Results generated with Lowry’s method are more similar to the data obtained from the sum of the amino acid residues which is considered the most reliable way of determining the actual protein content. Protein values obtained with BSA as a protein standard were closer to those calculated from the sum of amino acid residues, suggesting that the use of BSA is more suitable for the Lowry method. The procedures proposed here can contribute to better results, since protein is extracted efficiently and potential interference from compounds such as pigments, lipids, phenolics, small peptides and free amino acids is eliminated.
459 Acknowledgments We are indebted to FAPERJ (Foundation for Research Support of Rio de Janeiro State, grant E26.170.041/98) for the financial support to this study. Special acknowledgements are due to Dr. Yocie Yoneshigue-Valentin (Universidade Federal do Rio de Janeiro) and Dr. Carlos Logullo de Oliveira (Universidade Estadual do Norte Fluminense) for offering us laboratory facilities to perform this study. We thank Dr Ursula M. Lanfer Marquez (Universidade de S˜ao Paulo) for her support in the amino acid analysis. E.B. acknowledges CAPES and S.O.L. acknowledges FAPERJ and CNPq for providing them research fellowships.
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