The Mitochondrial Protein - Lab Report Example

Spectrophotometric assays of mitochondrial protein Proteins are one of the most important molecules in any organism and therefore, determination of protein concentration is very important. In this work, concentration of protein in bovine serum albumin samples has been determined using three different spectrophotometric methods – Lowry method, Bradford method and Warburg-Christian method. The results of the experiments are presented and discussed in this paper. Introduction: Proteins are very important building block of all the organisms. They are synthesized by RNA or ribonucleic acids. They serve many important functions in the body. Our muscles are made of proteins and we derive our ability to actuate by our muscles only. It is therefore, not surprising that concentration of proteins is one of the most important biochemical parameters while studying biochemistry or biomedical sciences. Concentration of any substance is represented by normalizing its mass by the sample size. For an example concentration of sugar in water solution can be expressed as gm/lit of the solution. It may as well be expressed in terms of molar mass like concentration of normal salt in a solution can be expressed as mol of NaCl per litre of solution. Many times it must be expressed in terms of some proxy of biomass like a mitochondrial enzyme activity might be expressed as lmolmin-1.mg-1 (mitochondrial protein) or as molmin-1.mg-1 (tissue protein). Concentration of protein can be measured in different ways. Some of the prominent methods employed for measurement of the concentration of protein in a suspension involve are based on spectrophotometry. What is done essentially is that the absorbance of a particular wavelength of light is measured and this absorbance is dependent on the concentration of the absorbing species, protein in this case. In fact spectrophotometry is a technique widely used for concentration determination of many other species in chemistry as well. These techniques are also known as colorimetry. Three different colorimetric techniques used for determination of protein assays will be used in this experiment for determination of the assays of mitochondrial protein. 1. Lowry method: In this method there are two reactions used to prepare the sample for colorimetry. The first reaction is biuret reaction, in which the alkaline cupric tartarate reagent complexes with the peptide bonds of the protein. Subsequently the resulting solution is reduces the Folin and Ciocalteu’s phenol reagent, which yields purple colour. Absorbance of this coloured solution is measured for a wavelength of 600 nm. The protein concentration is then determined from a calibration curve. 2. Bradford method In this method, the Bradford reagent is used which is a dye, Brilliant Blue G. This forms a complex between the dye and the protein in the solution. This protein – dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorbance is proportional to the protein concentration in the solution and again the protein concentration is determined using spectrophotometry and the calibration curve. 3. Absorption of 280 nm light This is based on absorption of ultraviolet light of 280 nm wavelength. This wavelength is absorbed by the aromatic amino acids present in proteins. This is again based on spectrophotometry and use of the calibration curve. As all these techniques are based on spectrophotometry, therefore, it is relevant to briefly discuss the principle of spectrophotometry. Spectrophotometry: When a light beam passes through a solution a part of the light beam is absorbed by the solution, the degree of absorption depends on the concentration of chemicals in the solution and the length of the path travelled by the light beam through the solution and the wavelength of light. To negate the effect of the wavelength a monochromatic light source is used and to negate the effect of path length a solution holder of fixed diameter is used. Thus there is a fixed quantitative relationship between the concentration of the solution and the degree of absorption by the solution. This relationship is known as Beer’s law and is represented as (http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html): It = I0e-kc Where, I0 is intensity of the incident light, It is the intensity of the transmitted light, k is the absorption coefficient and c is concentration. Taking the logarithm of both sides in the above equation yields kc = ln(I0/It) Where, kc is optical density of the solution and this is directly proportional to the concentration of the solution and is negative of the ln of absorbance of the light. Materials and Equipments: In this experiment a spectrometer and Quartz glass cuvettes were used. To prepare the solutions for colorimetric analysis 10 mg.ml-1 of bovine serum albumin, Lowry reagent (Alkaline cupric tartarate), Folin and Ciocalteu reagent (prepared for this experiment) and Bradford reagent was used. Experimental Method: The stock protein standard solution was diluted using distilled water to make following standard protein solutions: 20ml1.0mg.ml-1, 20ml0.8mg.ml-1, 20ml0.6mg.ml-1, 20ml0.4mg.ml-1, 20ml0.3mg.ml-1, 20ml0.2mg.ml-1 and 20ml0.1mg.ml-1. Lowry Method (http://www.ruf.rice.edu/~bioslabs/methods/protein/lowry.html): 1. The Lowry reagent was used as it is; while the Folin and Ciocalteu’s phenol reagent was diluted by adding distilled water in 1:5 by volume to make the working solution. 2. Standard tubes were prepared, in triplicate by using the protein standard solution. 3. One of the tubes was marked blank and 1.0 ml distilled water was filled into it. The mitochondrial sample was added in another test tube and it was marked for identification. 4. 1.0 ml of Lowry reagent solution was added to the standard, blank and sample tunes and it was mixed thoroughly and allowed to settle for 20 minutes. 5. 0.5 ml of the Folin and Ciocalteu’s phenol reagent was added with rapid immediate mixing and the solution was left for 30 minutes for colour to develop. 6. The solutions were transferred to cuvettes and the absorbance of the standards and sample tubes versus the blank was measured at 600 nm. Measurements were completed within 30 minutes. 7. Absorbance of the standards was plotted against their concentration and the calibration curve was prepared. 8. Concentration of the sample was measured from the calibration curve. Bradford Method (http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html): 1. 0.1 ml of the standards and sample was added into respective test tubes. One test tube was kept blank. 2. 3.0 ml of the Bradford reagent was added in all the test tubes, gently mixed and incubated for ~ 30 minutes. 3. The solutions were then transferred in respective cuvettes and absorbance was measured at 595 nm. The measurements were completed within 60 minutes. 4. Standard curve was made and concentration of the sample was determined from the standard curve. Warburg-Christian method: 1. The standards and the diluted sample were taken into cuvettes. 2. Absorbance at 280 nm was measured and recorded. 3. Standard calibration curve was prepared and concentration of the sample was determined from the calibration curve. Results and Discussion: Absorbance of the standards and the diluted samples as per Lowry method is presented in the table 1, below: Table 1: Absorbance of the standards and the diluted samples S T A N D A R D S Concentration mg/ml Amount mg Absorbance at 600 nm Reading 1 Reading 2 Average 0.0 0.0 0.020 0.035 0.028 0.1 0.1 0.090 0.096 0.093 0.2 0.2 0.135 0.142 0.138 0.3 0.3 0.210 0.225 0.218 0.4 0.4 0.282 0.294 0.288 0.6 0.6 0.385 0.392 0.388 0.8 0.8 0.520 0.520 0.520 1.0 1.0 0.641 0.646 0.644 Mitochondrial Suspension 1:100 dilution 0.358 0.364 0.361 Mitochondrial Suspension 1:200 dilution 0.184 0.179 0.182 The calibration curve is presented in figure 1, below: The calibration curve gives the following equation for the relationship between absorbance (y) and concentration (x in mg/ml): y = 0.6147x + 0.0284 Using this equation concentration of 100 times diluted solution (absorbance y = 0.361) will be 0.361 = 0.6147x + 0.0284 or, x = 0.54107 mg /ml As this suspension is 100 times diluted, therefore, Actual concentration will be 100x or 54.11 mg/ml. Similarly, using absorbance of 200 times diluted solution Actual concentration will be 200x or ~ 50.0 mg/ml. Absorbance of the standards and the diluted samples as per Bradford method is presented in the table 2, below: Table 2: Absorbance of the standards and the diluted samples S T A N D A R D S Concentration mg/ml Amount mg Absorbance at 600 nm Reading 1 Reading 2 Average 0.0 0.0 0.54 0.55 0.545 0.1 0.1 0.61 0.605 0.605 0.2 0.2 0.678 0.684 0.681 0.3 0.3 0.72 0.738 0.729 0.4 0.4 0.775 0.792 0.784 0.6 0.6 0.91 0.89 0.90 0.8 0.8 1.09 1.085 1.088 1.0 1.0 1.16 1.162 1.161 Mitochondrial Suspension 1:100 dilution 0.72 0.725 0.722 Mitochondrial Suspension 1:200 dilution 0.63 0.638 0.634 The calibration curve is presented in figure 1, below: The calibration curve gives the following equation for the relationship between absorbance (y) and concentration (x in mg/ml): y = 0.6326x + 0.5428 Using this equation concentration of 100 times diluted solution (absorbance y = 0.722) will be 0.722 = 0.6326x + 0.5428 or, x = 0.2833 mg /ml As this suspension is 100 times diluted, therefore, Actual concentration will be 100x or 28.33 mg/ml. Similarly, using absorbance of 200 times diluted solution Actual concentration will be 200x or ~ 28.83 mg/ml. Absorbance of the standards and the diluted samples as per Warburg-Christian method is presented in the table 3, below: Table 3: Absorbance of the standards and the diluted samples S T A N D A R D S Concentration mg/ml Amount mg Absorbance at 600 nm 0.0 0.0 0.041 0.1 0.1 0.048 0.2 0.2 0.058 0.3 0.3 0.065 0.4 0.4 0.07 0.6 0.6 0.082 0.8 0.8 0.098 1.0 1.0 0.112 Mitochondrial Suspension 1:100 dilution 0.082 Mitochondrial Suspension 1:200 dilution 0.065 The calibration curve is presented in figure 3, below: The absorbance of the diluted samples matches with those of the standard samples. Therefore, protein concentration in the samples can be easily determined and this comes to be 60 mg/ml. Conclusions: All the three methods yielded a linear curve; this is evident from very high value of R2 for all the three curves. R2 is a measure of correlation between the two related variables. Its value lies between 0 and 1; values near zero imply poor or no correlation while the values near 1 imply strong correlation. Therefore, it can be said that in all the three methods, there is strong linear correlation between absorbance and the concentration. Sensitivity of a method can be inferred from the slope of the calibration curve; higher is the slope of the calibration curve more sensitive is the method. Therefore, it can be said that Lowry and Bradford methods are equally sensitive and much more sensitive than Warburg-Christian method. The different methods have yielded different concentration of protein in the same sample. This could be due to different degree of interaction of the proteins with the reagents used in different methods and explanation of this discrepancy needs more work to be done. At the outset it can be claimed that all the three methods are equally capable of determining assay of protein in a sample. References: Hartree-Lowry and Modified Lowry Protein Assays http://www.ruf.rice.edu/~bioslabs/methods/protein/lowry.html retrieved on October 22, 2008 Bradford protein assay from http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html. retrieved on October 22, 2008. Spectrophotometry Principles of Spectrophotometry (http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html) retrieved on October 22, 2008 Read More