Two absorption spectroscopy assays, a colorimetric assay and a spectrophotometric assay, were performed to calculate the approximate calmodulin concentration in protein samples collected from a previous experiment. For the colorimetric assay, the method developed by Bradford was employed using coomassie blue dye and BSA, bovine serum albumin, as a standard. From this standard, a line was charted directly from the absorbance values of the BSA to their corresponding known concentrations. An R2 line was then plotted from these. According to the BSA standard analyzed by a microplate spectrophotometer, calmodulin was found to be present at concentration range of 3.25µg/mL - 527µg/mL with an average concentration of 176µg/mL. The spectrophotometric assay utilized an Ultra Spec 4000 spectrophotometer to scan the protein samples between the wavelengths of 190 to 340nm. The absorption maxima produced at the 280nm wavelength detected calmodulin at a concentration of 308µg/mL using a direct value of 1µg/mL ~ A280. Experimental error was present from contamination of DNA was found through the spectrophotometric analysis and through bad pipetting technique during dilutions in the Bradford assay.

Absorption spectroscopy is a common method for finding the concentration of proteins or protein complexes in a solution (1). Proteins absorb light at specific wavelengths and can be defined by the equation A? = log (I0/I). This equations states that an absorbance at a specific wavelength, A?, is equal to the log of the ratio of incident light intensity, I0, to transmitted light intensity, I (4). This can be better understood when looking at Figure 1 below. This absorbance, A?, is directly proportional to the concentration of the protein in solution (1). To identify this concentration, an equation known as the Beer-Lambert Law is used (1,2).

In this study, the absorbance is measured with an ultraviolet-visible spectrophotometer. As light if produced by two bulbs, one producing ultraviolet light and the other visible light, the light goes through a monochromator(1,4). The monochromator reduces the light to one single wavelength which is then transmitted through the sample in a cuvette(1,4). The cuvette for ultraviolet wavelength readings must be made of quartz since the other options, plastic or glass, absorbs light at this wavelength (1,2). The light is then transmitted through the sample in the cuvette to a sample detector(4). In front of the cuvette, a reference detector records the amount of light entering into the sample(1). The absorbance is then calculated from these two light intensities and charted in a digital readout (1) as shown in Figure 2.

Two absorbance spectroscopy assays were used in our study, a colorimetric assay and a spectrophotometric assay.
The colorimetric assay uses a dye bound to the protein to measure absorbtivity. Coomassie blue is a hydrophobic dye used in the Bradford method to bind to the hydrophobic portions of proteins. Coomassie blue, in its protonated form, has a maximum absorption of light at 465nm. However when placed in a dilute acidic solution its anionic form, it binds preferentially to argenine residues in proteins and the complex absorbs light maximally at 595nm (1,2). The amount of dye bound can then be detected by a spectrophotometer. BSA, or bovine serum albumin, was used as a protein concentration standard in this study. Since the amount of absorbance for BSA is known, it produces a standard curve on a digital readout from which unknown protein sample concentrations can be measured.

A more direct method for analyzing protein concentration is the ultraviolet spectrophotometic assay (3). A spectrophotometer transmits a light over a set wavelength range and the absorbance maxima on a digital readout can be observed. Trypophan, tyrosine, histadine, and phenylalanine residues contain aromatic rings that absorb the greatest amount of light at 280nm (2). At 280nm, the p electrons in the ring structures absorb energy from the light and are excited to the p* state (3). The decrease in light transmitted is then recorded and produces an absorption maxima, or peak, on a digital readout. In this study we used a concentration standard with 1 A280 being equal to 1mg of protein per mL of solution. An assumption made in this method is that the protein contains an average number of tryptophan and tyrosine residues (2).

MATERIALS AND EQUIPMENT: BIORAD Benchmark Plus microplate spectrophotometer, Microplate Manager Ver. 5.1, 12X8 microtiter plate, Ultra Spec 4000 spectrophotometer (Phamacia Biotech) CHEMICALS AND REAGENTS: BSA (Omnipur) # 1979A79, BIORAD Bradford Reagent (Sigma) #47115980, Tris-HCl (Promega) #125213, NaCl (Fisher Scientific), EGTA (0.2M)

A stock solution (24.2% BSA (v/v) in water) was prepared. From this stock solution, serial dilutions were then made 1:1 with water to 1/16th the original stock concentration. The wells for standard solutions were then filled in duplicate with the serial dilutions and Bradford reagent [25% BIORAD Bradford Reagent (v/v in water)] in a 3:1 ratio respectively as shown in Table 1 below. Dilutions of the protein of 1/7, 1/10, 1/14, 1/22, and 1/25 in buffer (27.5mM Tris-HCl, .55M NaCl, 2.75mM EGTA in di water) were prepared and pipetted in duplicate into the microtiter plate as shown in Table 1 below..

The microtiter plates were then analyzed in a microplate spectrophotometer and analyzed with Microplate Manager. The raw data was then viewed to see if the concentrations of the protein samples fell within the range of the standards and the procedure repeated with changed protein dilutions if not.

A quartz cuvette was obtained and filled with pure BSA. The cuvette was then put into the sample holder of the Ultra Spec 4000 spectrophotometer. The sample was analyzed with a wavelength range of 190nm to 300nm and analyzed.
The cuvette was then cleaned with 70% ethanol, filled with a 1:2 dilution of the BSA, and reanalyzed as before. This analysis was then repeated with a 1:4 dilution of the protein sample.

Several of the absorbance values for the protein sample dilutions in the Bradford assay were outside the range of the standard BSA values. These values are shown in red in Table 1 below and were not used in plotting absorbance values in Figure 3. Only the figures in black were used for calculating the concentration range of calmodulin in the protein sample. For calculation of the Protein Sample Dilutions concentration values in Table 2, the equation derived from the R2 line in Figure 3 was used with the unknown absorbance values substituted for “y” and the equation solved for “x”. From these concentrations of the diluted protein samples, the value was multiplied by the inverse of the dilution factor in Chart 1 to find the original concentration. Although these values should be similar, a range was found between 3.25µg/mL and 527.5µg/mL.

Standard BSA:Protein Sample Dilutions

Diluting the BSA sample 1:2, the absorbance maxima again produced a peak around 280nm with half the peak value of undiluted bsa. This shows a direct correlation between absorbance value and protein concentration.

In the spectrophotometer readout on the unknown protein sample, the peak shifted to the left with a maxima at 260nm at an absorbance value of 0.71. At 280nm, an absorbance of 0.38 was shown.

Calmodulin may have been present as was evidenced by an absorbance value of 0.38 at 280nm. However, since the absorbance value was shown to correspond directly to concentration values in the bsa spectrophotometric charts, it can be concluded that a contaminant with an absorbance value of 0.71 at 260nm was at least twice as concentrated as calmoduin. From Figure 2, it is seen that nucleic acids absorb light at 260nm and is mostly likely our contaminant. While the spectrophotometric analysis failed to provide any valid estimate of calmodulin concentration in our protein sample, the Bradford assay did give a range of possibilities. The unfortunate side to our range is the large possible concentration values between 3.25µg/mL and 535µg/mL. An average of these possible values is 176µg/mL.

To provide more precise information on the concentration of calmodulin in the protein sample, nucleic acids need to be removed and the lab techniques, such as pipetting, must be more accurate. One possible suggestion for more pipetting in the Bradford assay is to mix the dilutions in higher volumes, then add from these volumes into the sample wells. Since the volumes used were in the range of µg, the precision required for mixing solutions to a total volume of 1mL per well is extremely high and difficult. REFERENCES

1. http://tecn.rutgers.edu/bio301s/Lab%203-%20protein%20diagram.htm
2. Millinder S., Clack B. Chemistry 555 Laboratory Manual. pp 6,10,13. Stephen F. Austin State University Biotechnology Division, TX.
3. http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/uvvisab1.htm
4. Mathews C. K., Van Holde K. E., and Ahern K. G.. (2000) Biochemistry: Third Edition, pp. 477, 1151. Addison Wesley Longman, California.

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