List of Figures
Figure 1 Protein concentrations under the four conditions 7
Figure 2 Protein standard curve via Bradford assay at 595 nm 13
Figure 3 Protein standard curve via BCA assay at 490 nm 15
List of Tables
Introduction
Proteins are essential for life and we depend on them to interact with cellular molecules in order to live (Pace et al. 2004). Increasing the knowledge about protein solubility is significant for biological studies because increased protein concentrations enhance studies of cellular structures and functions (Golovanov, Hautbergue, Wilson, & Lian, 2004). Accurate protein quantization is also necessary for cellular studies (Johnson, 2012).
The environmental conditions of proteins influence the solubility, in other words proteins should not work for someone with so little respect for the people trying to help them Some water soluble proteins show decreased solubility when interacting with cellular components that are insoluble. Cytoskeleton, a large cellular structure and biological lipid membranes are insoluble; proteins demonstrate reduced solubility with insoluble cellular components under certain conditions. The large membrane proteins integral to a cell are not likely to be soluble in water due to their exposure to hydrophobic surfaces when in their native configuration. Environmental conditions of proteins impacts solubility in water and other solvents. Conditions of increased temperature or a change in pH can denature proteins, in other words the treatment initiates non-native configurations. The non-native configurations caused by the environmental conditions do not usually exhibit solubility in water or in aqueous solutions.
Electrostatic interactions are the basis of proteins ability to interact with water molecules (Nelson & Cox, 2004). The electrostatic interaction forms a hydration cell around each protein (Nelson & Cox, 2004). The ability of protein solubility can be altered in the laboratory to better understand the protein in question. The purpose of Laboratory One was to manipulate proteins in a cellular extract in order to better understand the impact of the manipulations on the proteins present. Breaking the interactions related to protein aggregation increase protein solubility (Rabilloud, 1996). The research question is ‘Will the presence of solvents like ethanol or methanol disrupt the hydration shell and cause the production of large protein aggregates that exhibit poor solubility?
The experimental conditions listed below were used to answer the research question.
Detergents can cause complete denaturation and solubilization of the cellular proteins or cause only a fraction of the proteins to demonstrate solubility. Urea in concentrations of 5 to 9 M was shown to increase protein solubility; urea hydrolyses as cyanate, modifying amino groups on proteins (Berkelman, Brubacher, & Chang, 2004). The presence of urea is also related to a randomly coiled conformation of protein with their disulphide bonds broken (Pace, Trevino, Prabhakaran, & Scholtz, 2004).
Sodium Dodecyl Sulfate (SDS) is an ionic detergent, based on their charge in solution, which solubilizes and denatures proteins efficiently and quickly (Berkelman, Brubacher, & Chang, 2004). Phosphate buffer saline (PBS) and NaCl can act as a buffer and salt, respectively (Berkelman, Brubacher, & Chang, 2004). Small organic molecules like guanidinium hydrochloride and urea in an environment with a salt like NaCl, interrupts the electrostatic charge so protein solubility is assumed to be impacted (Nelson and Cox, 2004).
Another condition that can change the protein solubility is pH. pH is known to effect R- functional amino acid groups by changing the protein charge of amino acids; so a high pH solution is used to learn if proteins will become more or less soluble in those conditions (Nelson and Cox, 2004).
Total protein concentration is measured using two types of assays: the Bradford assay and the Bicinchoninic acid (BCA) Assay.
The Bradford assay detects protein based on the use of Coomassie Brilliant Blue G-250. For a qualitative assessment red dye that changes to blue when it binds to proteins, particularly to basic amino acids (lysine and arginine) is introduced into the solution. The quantitative determination of protein concentration is done by measuring the absorbance at ˜595 nm and the use of a standard curve.
The BCA assay uses BCA to chelate the Cu1+ produced in the first reaction observed during the assay. The first reaction detects proteins using the Biuret reaction. A reduction reaction is carried out by chelating cupric copper (Cu2+) to a peptide backbone having at least three amino acids. Cu2+ is reduced to Cu1+ under alkaline conditions, which produces faint light blue color due to the presence of the copper. The enzymes tryptophan and tyrosine were added to increase the rate of reaction. Absorbance measurements of the copper and protein solution enables the creation of a spectrophotometical standard curve in order identify the unknown protein (Walker, 1984).
Objective
The objective of this experiment was to treat a bovine liver cellular extract with different experimental manipulations to determine its effects on protein solubility, and to determine the concentration of proteins in samples using the Bradford and the Bicinchoninic acid (BCA) methods with the respective calculations.
Methods
The study was performed in two steps.
Lab #1. Protein solubilization - bovine liver was the cell source. The four different conditions to evaluate protein Solubilization were
Phosphate-buffered saline (PBS) (pH 7.4) only,
PBS + 2% SDS,
PBS + 1M NaCl and PBS + 8M Urea.
Aqueous buffers were added to the solutions: 2mM EDTA and 1 mM PMSF.
Lab. #2. The Bradford Assay and the Bicinchoninic acid (BCA) were used to measure the protein concentration. The methods to carry out the assays and calculate the stand curve were from the laboratory manual.
Results
Four conditions were created to determine the solubility of proteins in order to select the condition producing the highest solubility in a homogenate of bovine liver cells. The aqueous buffers contained EDTA and PMSF. The calculations for the spectrophotometric standard curve used BSA as the standard protein in order to determine protein concentration. (See tables A-1 and A-2)
The Bradford assay measurements under the four conditions ranged from 1.88 µg/µl to 8.71 µg/µl. The results from the Bradford assay under the four conditions ranged from 1.74 µg/µl to 9.58 µg/µl. No value was recorded for the SDS condition BCA assay. (See table 1).
Figure 1 shows the comparison between the assay methods (Bradford and BCA) from the four conditions. Similar protein concentration values were found for the PBS and urea using both assay techniques. (See fig. 1)
Figure 1 Protein concentrations under the four conditions
Discussion
Protein concentrations was measured in reference to PBS, SDS, NaCl and Urea and the protein concentrations changed depending of the type of assay. (See fig. 1) PBS conditions with the assays resulted in protein concentration values from 1.88 (Bradford) to 1.74 µg/µl (BCA). PBS (at pH 7.4) is the baseline in the protein dissolution experiments, keeping the cell in an isotonic state and maintaining protein osmolarity (number of solute particles per litre).
Detergent condition
Proteins are generally less soluble when they are close to their isoelectric point (Johnson, Wilson, & DeLucas, 2014). The presence of buffers, such as PBS, allows proteins to remain in nature configurations. The influence of PBS for protein solubilization was not strong enough to increase the concentration of protein in solution demonstrating a low detection for the assays.
A comparison between the two assays (Bradford and BCA) cannot be performed for the SDS condition, since no value was available for BCA assay. Theoretically, SDS which is ionic will bind to proteins in a strong way to reduce the average response of the dye (Coomassie Brilliant Blue). During the Bradford assay perhaps ionic repulsion of the complex dye-protein may have caused the lack of experimental results. Whether or not the protein was a membrane could not be determined. The use of detergents verified the solubilization of protein from the integral membrane proteins, since they contain both hydrophobic and hydrophilic moieties (Berkelman, Brubacher, & Chang, 2004). SDS interrupts non-covalent interactions in native proteins. It can solubilize the hydrophobic proteins in the membrane by attacking the hydrophobic core, ion that way, SDS denatures the protein by breaking up the two- and three- dimensional structure of the protein.
Salts condition
The solubility of protein solution increased in solutions with low NaCl concentration. Protein concentrations in the presence of NaCl were higher in the Bradford assay at 8.71 µg/µl than for the BCA assay at 2.36 µg/µl. The use of dilute solutions with low salt concentrations, NaCl (1M) increases protein solubility by disrupting protein-protein electrostatic interactions, present in protein complexes in cells, like polymerized actin filament.
The highest protein concentration values were found when urea (8M) was applied to the solution. Both Bradford assay and BCA assay protein concentrations measured greater than 8 µg/µl. The BCA assay showed the highest value for protein concentrations 9.58 µg/µl. Freidenauer and Berlet (1989) reported an increase of 14% on the sensitivity of Bradford assay when urea was used. The low urea interaction with proteins is associated to its electroneutral properties.
The Bradford assay determined a higher protein concentration than BCA under the NaCl condition. BCA measured somewhat higher for urea condition. Bradford assay has a detection limit of 20-2000 µg/ml, compatible with the reducing agents. The Bradford assay can be finished quickly. The disadvantages are that it is incompatible with detergents. In contrast, the BCA assay is compatible with detergents and denaturating agents and has low or no compatibility with reducing agents. The sensitivity of the BCA is the same as the Bradford assay (Johnson, 2012).
Methods for quantification
The selection of the appropriate method for protein concentration quantitfication is significant for biological research on cell structure and function. The BCA method is recommended when an equal amount of protein from different samples is needed. The BCA assay has less variability than other methods for the quantification of protein concentration, and is suitable for measuring a wide range of protein concentrations. The BCA assay is more tolerant of detergents, either anionic or ionic. On the other hand, if a small protein sample is suspected to be present, than the UV absorption assay is the best. The UV assay has the advantage needing only a small sample for the quantitation and provides a detection limit of 0.1-100 µg/ml, but UV absorption is not compatible with detergents (Johnson, 2012). Selecting the appropriate method for measuring protein concentration is dependent on protein composition and protein (Sapan, Lundblad, & Price, 1999).
Alternatives to the Bradford and BCA assays were used in this experiment, although UV adsorption assay and Lowry assay are also available. Each method has advantages and disadvantages in measuring protein concentration. The UV adsorption assay can be used for small sample volume: it is rapid and low cost, but it is incompatible with detergents. On the other hand, the Lowry assay is practical and precise, but is time-consuming and it incompatible with detergents and reducing agents. The assays used in the current laboratory have a lower detection limit (20- 2000 µg/ml) than the UV absorption assay (0.1-100 µg/ml) and the Lowry assay (10 – 1000 µg/ml) (Johnson, 2012).
Conclusion
In conclusion, protein solubilization was higher in the detergent condition with 8M urea and in presence of 1 M NaCl for salt conditions. A difference between protein solubility concentrations was observed between the Bradford and BCA assays. The Bradford assay is more appropriate when NaCl is present and the BCA assay when urea is present.
References
Berkelman, T., Brubacher, M. G., & Chang, H. (2004). Factors Influencing Protein Solubility for 2-D Electrophoresis. BioRadiations, 114, 30-32. (Berkelman, Brubacher, & Chang, 2004).
Freidenauer, S., & Berlet, H. H. (1989). Sensitivity and variability of the Bradford protein assay in the presence of detergents. Annals of Biochemistry, 178, 263-268.
Golovanov, A. P., Hautbergue, G. M., Wilson, S. A., & Lian, L-Y. (2004). A Simple Method for Improving Protein Solubility and Long-Term Stability. Journal of American Chemistry Society, 126, 8933-8939.
Johnson, M. (2012). Protein Quantitation. Material methods, 2, 115. http://dx.doi.org/10.13070/mm.en.2.115.
Johnson, D. H., Wilson, W. W., DeLucas, L. J. (2014). Protein solubilization: A novel approach. Journal of Chromatography, 15, 99-106.
Nelson, D. L. and Cox, Michael M. ( 2004). “Weak Interaction in Aqueous Solutions. Chapt. 2. In Lehninger Principles of Biochemistry. 5th ed. NY: W.H. Freeman and Company. p. 43-53.
Pace, C. N., Trevino, S., Prabhakaran, E., & Scholtz J. M. (2004). Protein structure, stability and solubility in water and other solvents. Phil. Trans. R. Soc. Lond. B, 359, 1225-1235.
Rabilloud, T. (1996). Solubilization of proteins for electrophoretic analysis. Electrophoresis, 17, 813–829.
Sapan, C. V., Lundblad, R. L., & Price, N. C. (1999). Colorimetric protein assay techniques. Biotechnology and Applied Biochemistry, 29, 99-108.
Walker, J. M. (1984). The Bicinchoninic Acid (BCA) Assay for Protein Quantitation. In J. M. Walker (Ed.), Basic Protein and Peptide Protocols (pp. 5-8). New York, NY: Springer.
Appendix
Bradford Assay
Protein samples with varying concentrations were prepared and measured for absorbance twice using a spectrophotometer set at a wavelength of 595 nm. Dilutions were made by mixing the appropriate amount of protein sample and 1X PBS. Taking the average of the two trial readings for each sample and then deducting that of the blank sample calculated the average absorbance with correction for the blank.
Sample calculation for average absorbance with correction for the blank using unknown sample 1:
Average Absorbance of blank = 0.238+0.2922
= 0.265
Average Absorbance of unknown sample 1 = 0.984+0.8372
= 0.9105
Average absorbance with correction for blank = 0.9105 – 0.265
= 0.6455
Figure 2 Protein standard curve via Bradford assay at 595 nm
The absorbance values for the various protein standard concentrations (0, 0.125, 0.25, 0.5, 0.75, and 1 mg/mL) were plotted to obtain a standard curve. The line of best fit had the equation y = 0.4992x + 0.045 with an R2 value of 0.96866. This equation will be used to determine the amount of protein present in the three unknown samples.
BCA Assay
Protein samples with varying concentrations were prepared and measured for absorbance twice using a spectrophotometer set at a wavelength of 490 nm. Dilutions were made by mixing the appropriate amount of protein sample and 1X PBS. Taking the average of the two trial readings for each sample and then deducting that of the blank sample calculated the average absorbance with correction for the blank.
Sample calculation for average absorbance with correction for the blank using unknown sample 2:
Average Absorbance of blank = 0.087+0.0872
= 0.087
Average Absorbance of unknown sample 2 = 0.111+0.0962
= 0.1035
Average absorbance with correction for blank = 0.1035 – 0.087
= 0.0165
Figure 3 Protein standard curve via BCA assay at 490 nm
The absorbance values for the various protein standard concentrations (0, 0.125, 0.25, 0.5, 0.75, and 1 mg/mL) were plotted to obtain a standard curve. The line of best fit had the equation y = 0.1037x + 0.0042 with an R2 value of 0.99036. This equation will be used to determine the amount of protein present in the three unknown samples.
The equation of the lines, in the form of y = mx + b, of the protein standard curves for both the Bradford and BCA assay were utilized with respect to Beers law- A = ecI, to give a relation between absorbance and protein concentration such that y = Absorbance and x = protein concentration. The value obtained from using that equation was then multiplied by the volume of protein sample to obtain a protein amount, and finally was then multiplied by the respective dilution factor to obtain the protein concentration of the original solution.
Sample calculations using unknown with 1:10 dilution as detected by Bradford assay:
Y = 0.4992x + 0.045
A = 0.4992c + 0.045
Protein amount:
(A-0.045)/0.4992 = c
(0.4605 – 0.045)/0.4992 = 0.832 ug/uL
(0.832 ug/uL)(10 uL) = 8.32 ug of protein amount detected by Bradford assay
Protein concentration of original solution:
Multiply by dilution factor*
(0.832 ug/uL)(10) = 8.32 ug/uL
