8. SUMMARY IN SPANISH / RESUMEN EN ESPAÑOL
8.3 Conclusiones
Protein-protein variations in assay results were similar to those observed with the batch assay: gelatin < BSA < chymotrypsinogen < RNA < insulin. The within-day precision of analysis was 1.04±2.99% and day-to-day precision was 1.62±14%, depending on the nature and concentration of the protein analyzed.
8. APPLICATIONS OF THE BCA ASSAY TO FOOD PROTEIN ANALYSIS
There are only a small number of reports describing the use of the BCA assay for food protein analysis. No doubt, the numbers of applications will
increase in the near future.* Applications reported in the published literature are discussed next.
8.1. Solid-Phase Analysis of Cereal Proteins
Chan and Wasserman (44) determined protein in corn meal ¯our.
Commercial corn meal samples and/or zein (2±7 mg) were placed in microcentrifuge tubes. The BCA working reagent (1 mL) was added and the mixture was incubated with intermittent shaking at 378C for 30 minutes.
Samples were cooled over an ice bath for 5 minutes and particulate material removed by centrifuging (13,000g;10 minutes). Thereafter, 0.2 mL of supernatant was diluted in 1 mL of BCA reagent A (see Method 1 for the reagent composition) and A562 readings were recorded. The BSA (50±
400 mg) was used as the standard protein while method calibration involved Kjeldahl analysis.
Fig. 2 shows generally good agreement between Kjeldahl and BCA results. However, the former technique gave higher values for protein than the BCA assay. The discrepancy between Kjeldahl and BCA assays was
FIGURE 2 Correlation between Kjeldahl protein and BCA assay of corn meal protein and samples of zein. (Drawn from Ref. 44.)
* From personal experience, use of the commercially available reagent is underreported.
ascribed to the presence of NPN. It is also likely that BSA is not a satisfactory standard for corn protein analysis.
8.2. Analysis of Forage Plant Leaf Proteins
Some 23 forage plant (leaf) samples were analyzed using the BCA assay by Messman and Weiss (45). These included alfalfa (fresh, wilted, hay, silage, leaves), crown vetch (fresh, wilted, silage), spinach (fresh), perennial ryegrass (fresh), orchard grass (fresh, wilted), corn plant (silage), and peal millet (fresh). For sample pretreatment, leaves were lyophilized and ground using a 1-mm Wiley mill. The resulting powders were extracted with borate-phosphate buffer (0.1 M ionic strength) containing SDS (1% w/w). Sonicat-ing the suspension of leaf powder for up to 2 minutes facilitated protein extraction. In most cases, protein recovery was 85%. The protein extracts were analyzed with the BCA and Kjeldahl methods (N 6 6.25).
The BCA method gave unreliable estimates of leaf proteins. There was poor agreement between BCA and Kjeldhal results. Leaf samples contain numerous interfering compounds (plant pigments, peptides, sulfhydryl compounds, and phenol derivatives) that can interfere with both the BCA and Kjeldahl methods. Attempts to circumvent interferences using the DOC-TCA procedure were not successful. The yield of leaf protein recovered by precipitation with DOC-TCA ranged from 40 to 80%. Protein recovery by cold-acetone precipitation was not signi®cantly higher.
8.3. Analysis of Seed Proteins in the Presence of Phenolic Compounds
Salt-soluble proteins from soybean, tamarind, ragi, jack fruit, mango kernel, and sorghum were analyzed by Kamath and Pattabiraman (29). Whole meals were extracted with 0.3 M NaCl (buffered with 20 mM phosphate buffer, pH 6.9). Protein extracts were then analyzed using the BCA, Bradford, and Lowry assays. A range of pure proteins (BSA, casein, chymotrypsinogen, lysozyme, myoglobin, trypsin, zein) were also analyzed.
The BCA, Bradford, and Lowry assays showed differences in protein-protein variations in color yield. Apparently, endogenous seed compounds affected the results. High responses were obtained with sorghum, mango kernel, and other samples known to have high concentrations of total phenol. The BCA reagent was more sensitive to phenolic substances than to protein. On a scale of 1.0 for BSA, the color yields from a range of phenolic compounds were pyrogallic acid (86), gallic acid (2.1), pyrocatechol (106.0), tannic acid (9.3), and phenol (0.8). The BCA response to protein and phenolic substances was additive. There was a linear response between A567
values and the concentration of phenol. Most seed contained comparable amounts of protein and phenolic compounds. Therefore, the BCA response to these systems is likely to be due to the phenol. The BCA analysis of soybean protein was error free owing to the low concentrations of phenolic compounds in this seed.
8.4. Identi®cation of High-Lysine Cereals Using the BCA Assay
The A562 readings for ribonuclease, chymotrypsinogen, insulin, and BSA apparently showed a high degree of correlation with the number of lysine residues in each protein (R 0.99; P <.01). Therefore, the BCA assay response was determined for four high-lysine mutants of barley (Hordeum vulgare) and four normal-lysine variants. Whole meals were extracted with 4% SDS solution in 0.15 M Tris-HCl buffer (pH 7) at 608C for 20 minutes.
The extracts were diluted three fold with distilled water before analysis using the BCA assay at 258C for 2 hours.
There was a signi®cant correlation between BCA protein assay results and lysine content expressed per weight of barley meal (R 0.974, P <.001) (46). Available lysine was also determined using the TNBS (trinitrobenze-nesulfonic acid) method of Kakade and Liener (47) (see Chapter 12). A signi®cant correlation was also observed between BCA results and sample protein content as determined by the Kjeldhal method or by UV absorbance. As a corollary, the sensitivity of the BCA assay for a range of synthetic polyamino acids increased in the order Asp & His < Glu < Arg
Lys.
8.5. Determination of Plant Protein in the Presence of Reagents for Electrophoresis
Extraction buffers for SDS-PAGE, two-dimensional (2D) electrophoresis, and isoelectric focusing of plant proteins are incompatible with many protein assays. The 2D electrophoresis samples may contain 9 M urea, 2,2ME, SDS, and ampholyte. Orr et al. (48) assayed such samples accurately using the BCA assay.
8.6. Analysis of Animal Carcass Total Protein
Analysis of protein nutrient quality sometimes requires a quantitative determination of total carcass nitrogen (see Chapter 12). Brooks et al. (49) assayed whole-body nitrogen for male Sprague-Dawley rats using serveral methods including the BCA assay. Their approach should be applicable for
other animal carcasses with roughly similar fat content. This work might be usefully compared with that of Toten and Whitaker (50) described in Chapter 2. Carcasses were passed through a Horbart meat grinder (model 4812) once. The skin (including hair) was cut into pieces with scissors and added to the meat. Homogenizing in a Waring blender and a Brinkman polytron homogenizer further reduced the meat particle size. Finally, 1 g of protein was solubilized by homogenizing with (a) water, (b) 5% SDS dissolved in 0.5 M sodium hydroxide, or (c) 6 M guanidine hydrochloride solution.
Samples were analyzed by the BCA method, biuret assay, or Bradford assay. Protein extracts were also analyzed by three so-called absolute methods, i.e., quantitative amino acid analysis, protein hydrolysis followed by ninhydrin analysis, and Kjeldahl analysis. Quantitative amino acid analysis yields a value of 16.3 (+0.5) g protein/100 g sample (n 15 replicates). However, Kjeldahl results (N 6 6.25) were 34% higher than expected. Accurate results were obtained by using Fk 5.51. The ninhydrin assay results depended on the choice of amino acid standard. The BCA and biuret methods were not adversely affected by the presence of SDS or guanidine hydrochloride. The Bradford procedure was unusable in the presence of SDS (Chapters 6 and 7). These results are summarized in the following.
Apparently, SDS-NaOH was the most ef®cient protein extraction solvent tested. Thereafter, the accuracy of results varied in the order biuret
> BCA >> Bradford. The performance of the BCA assay may be better than indicated from results in Table 8. First, the BCA assay was not adversely affected by SDS-NaOH or GnHCl when serum albumin was TABLE8 Effect of Extraction Solvent on the Apparent Carcass Protein
Concentrationa
Assay method Extraction solvent [Protein] (g/100 g sample)
BCA Water 2.05
aWhole-body protein concentration was 16.3% by quantitative amino acid analysis.
employed as the standard protein. Inadequate explanation was given for the ability of the BCA assay to detect only 75% of the carcass protein.
Calibration graphs for the BCA are nonlinear. However, a hyperbolic function was not applied by the authors although such an equation was
®tted to results from the Bradford assay. There are good prospects that the BCA method can be adapted for animal protein analysis, perhaps with SDS-NaOH as the extraction solvent.
8.7. Analysis of Proteins from Freshwater Algae
There were several sources of error during attempts to analyze proteins from freshwater algae. Meijer and Wijffels (51) noted that the ef®ciency of protein extraction from cells was variable. Attempts to facilitate extraction using chemical means led to interferences with the Bradford and BCA protein assays. Proteins could also undergo severe damage under harsh extraction conditions such as boiling with alkali. Such harsh treatments could lead to standard proteins being less representative of the sample.
Boiling Chlorella cells with 1 M NaOH for 30 minutes led to a recovery of between 3% (Bradford assay) and 14% (BCA assay) of the crude protein.
By comparison, extracting yeast cells under similar conditions produced protein recoveries between 76% (Bradford method) and 85% (BCA method).
When BSA standard protein was exposed to boiling 1 M NaOH for 30 minutes, there was 32% (Bradford method) or 85% (BCA assay) of the response recorded for the untreated proteins. Apparently, chemical damage due to heating at high pH is only partly responsible for the poor assay results. Chlorella protein was ef®ciently extracted by sonicating 50-mL samples of fresh algae suspended in sodium phosphate buffer (25 mM) containing 1% SDS. After sonication for 0.5, 1, and 3 minutes, there was 36, 80, and 104% recovery of crude protein as determined by the BCA assay. To avoid foaming and a rise in sample temperature, the period of sonication was divided into six 30-second intervals. In the presence of SDS, the Bradford assay could not be used.
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