Determination of Proteins Using Biuret and Lowry Assay Techniques - Essay Example

Determination Of Proteins Using Biuret And Lowry Assay Techniques Introduction and Aims The aim of protein assay is to understand how to draw a curve by using prepared solutions to establish the concentrations of unknown solutions of albumin using spectrophotometer. According to Senaratne, Park and Kim (2006), it helps to properly understand the difference between qualitative and quantitative testing. Further, the researcher is able to use the spectrophotometer by calibrating it to detrmine the absorbance of different levels of solutions (Peterson 1983). Protein assay is ctitical in analysis of agricultural, industrial and biotechnological products. As argued by Bama et al. (2010), it is also important for research especially in analysis of enzymes, lectins and antibodies. This paper covers two kinds of assays used in quantitateing total proteins. These includes the biuret and lowry techniques. Biuret assay, which is the least sensitive assay is among the colometric methods (Quereshi et al. 2010). It is mostly used due to its simplicity and less susceptibility to chemical interferance. The assay is dependent on polypeptide chelation of cupric iron in strong alkali. According to Mizuta et al. (2005), most biuret assays are used in samples containing 1 to 10mg protein/ml, which is then diluted five fold by other reagents to form deep purple color. On the other hand, lowry method is a colorimetric assay that is based on folin-ciocalteau reagent and cupric ions of phenolic groups (Muyonga, Cole & Duodu, 2004). It is a popular protein estimation procedure eventhough highly susceptible to discerning compounds that interfere and distort solubility of insoluble proteins. The assay start with copper ion complex that has peptide bonds, which are stabilized by tartrate in alkaline environment populary known as biuret chromophore. Gornall, Bardawill and David (1949) pointed out that biuret reaction is reduced under alkaline conditions of folin-ciocalteu reagent. Copper ions are used to enhance the reduction process. However, the principle chromogenic groups consist of the peptide linkages that reduced blue molybdotungstates, which catalyses polar amino acids, tyrosine and tryptophan. Nonetheless, the sensitivity of this test is based on protein composition and products of chemicals reaction resulting to heteropolymolybdenum blue solution after being in absorbance condition of approximately 750nm, a wavelenght that is out of range of many interfering colours (Layne, 1957). In these two experiments, the basic law of light absorption, populary know as Beer–Lambert law is used to explain the linear relationship between protein (collagen) concentration and absorbance (Cliché, Amiot & Avezard, 2003). The yield of collagen is calculated using the following linier equation: Y=(VxC)/ W Where; Yis the yield of collagen in mg/g Vis the volume of collagen solution in ml C is the concentration of the derived solution in mg/ml Wis the lyophilized weight in g Materials used: 1. Protein sample of unknown concentration 2. Standard BSA 3. Distilled water 4. Lowry reagent 5. Test tubes 6. Label 7. Test tube rack 8. Pipettes 9. Pipette bulb 10. Vortex mixer 11. Spectrophotometer 12. Cuvettes 13. Gelatin : 100µg cm-3 14. Globulin: 100µg cm-3 15. albumin: 200µg cm-3 Methods Lowry Technique: Procedure: 1. Prepare samples with up to 100 μg of protein 2. Label the 9 test tubes as (1 to 10) and place them in a test tube rack. 3. Add water as provided in the instructions. 4. Prepare diluted Folin-Ciocalteu reagent and the Assay Mix. 5. Add 0.5cm3 of the protein solution to tubes (2 to 10). 6. Add gelatin solution to tube 7 and 8 only. 7. Then add 2.5cm3 of solution D to each tube and mix well and leave the mixture at room temperature for approximately 10 minutes. 8. Add 0.25 cm3 of diluted Folin-Ciocalteu reagent and Vortex immediately. 9. After 10 minutes add 0.25cm3 of solution E to each tube but mix this immediately and leave at least for 20 minutes. 10. While waiting for 20 minutes to finish label all 10 cuvettes provided for you as (1 to 10). 11. After 20 minutes pure out the solution on each tube to each cuvette provided. 12. Zero the spectrophotometer against the reagent blank and Vortex the tubes to measure absorbance at 700 nm (or other appropriate wavelength). 13. Now record the results shown on the spectrophotometer and plot a standard curve by interpolation to determine the concentration of unknowns. Biuret Technique: Procedure 1. Pipette 2 cm3 of the biuret reagent into the first 6 test tubes. 2. Pipette 2 cm3 of the biuret blank reagent into the frst 6 test tubes. 3. Add 100 μL of the protein to six separate test tubes with the biuret reagent. 4. Add water as provided to all test tubes filled with biuret reagent. 5. Prepare100 μL of serum to all test tubes filled with biuret reagent. 6. Mix the contents of each tube thouroughly. 7. Prepare a blank-series to all separate test tubes with biuret blank reagent by adding 100 μL of each of the proteins. 8. Add 100 μL of serum to all test tubes filled with biuret blank reagent to prepare serum unknown. 9. Mix the contents of each test tube thoughroughly. 10. Maintain the cuvettes at room temperature for 15 minutes. 11. Using the reagent-series blank, zero the Specctrophotometer at 540 nm and measure the absorbance of the reagent series including the serum unknown. 12. Subtract the absorbance of the blank-series from the overall reagent series. 13. Plot a standard curve of the resulting values of both absorbance and concentration to determine the concentration of the unknown. Results: Lowry technique: According to Sadowska et al. (2003), a linear relationship is established between x-axis (absorbance) and y-axis protein (albumin). This indicates that a good linier relationship is only attainable within a given range of albumin concentrations. The curve below is instrumental in monitoring concentration of albumin along the extration process. The stratight line curve is described by the following equation: µg= -0.0675+168*A660 with A giving the absorbance rate. Figure 1: Linier relationship between absorption and protein concentration- Lowryy technique Biuret technique: In this experiment, every concentration has a varied absorbance (Liu, Li & Guo 2007). The below curve is plotted using the points of concentrations and absorbencies. A line is developed, which indicates the points of concentration on the x-axis. Figure 2: Linier relationship between absorption and protein concentration- Biuret technique 1 2 3 4 5 6 Protein concentration (mg 0.5cm-3) Discussion Lowry technique: Accordingto Kołodziejska, Sikorski and Niecikowska (1999), the Lowry method is based on two separate reactions. The first comprises formation of amide bonds with copper ion complex resulting into a reduced copper in alkaline solutions. Second is the reduction of Folin-Ciocalteu reagent by tryptophan and tyrosine residues. The reduced Folin-Ciocalteu reagent is blue in colour and, therefore, detectable using a spectrophotometer ranging from 500-750 nm. However, the Biuret reaction is not highly sensitive and therefore use of Folin-Ciocalteu reagent makes the assay highly sensitive than a mere Biuret reaction (Nalinanon et al. 2007). Nevertheless, the assay takes more time and is susceptible to many interfering compounds. Most of the interfering substances are commonly used as buffers in preparation of proteins (Lin & Liu 2006). Further, the Lowry assay is sensitive to variations in the quantities of tryptophan and tyrosine residues. According to Wang, Yang and Du (2009), if the protein being assayed has got unusual content of these compounds, a substitute should be used. Notably, the standard curve is linear in the 1 to 100 μg protein region. The absorbance is commonly achieved between 500 to 750 nm. Biuret technique: Protein determination through biuret technique is done using ultraviolet wavelengt of about 250-350nm (Aukkanit & Garnjanagoonchorn 2010). It shows the different spectrum with BSA and adbsorption. Although in this experiment the maximun absorption was observed at 270, 280nm was preferred as the most appropriate wavelenght. However, to obtain the highest sensitivity, the copper iron concentration was required to be about 0.2% eventhough the NaOH concentration of less than 10% was the most desirable. Tatrate salt was used to enhance solubility of copper ion. To arrive at different levels of concentration and absorption, interpolation method was used. We determined the slope by taking two points along the straight line and calculated the change in y over change in x. My trend line slope was 0.25 divided by 3. With these figures, we were able to come up with a linier equation; y=0.25/3x with y being the absorption rate and x, the concentration. By sustituting the absorbance for y, the level of concentration of unknown solution was determined at 4.586mg/ml. References Aukkanit, N. & Garnjanagoonchorn, W. (2010). Temperature effects on type I pepsin-solubilised collagen extraction from silver-line grunt skin and it’s in vitro fibril self-assembly, Journal of Science, Food & Agriculture, 90, pp. 2627-2632. 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