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Spectrophotometry Laboratory

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Spectrophotometry Laboratory CELL BIOLOGY SPECTROPHOTOMETRY Spectrophotometry Laboratory INTRODUCTION hemoglobin protein, these molecules are colorless in solution, that is, they do not absorb visible light. Spectrophotometry refers to the measurement of However, both nucleic acids and proteins absorb radiant energy as a function of wavelength. A radiation in the ultraviolet region of the electro- spectrophotometer consists of a monochromator and a photocell connected electrically to a meter or recorder. The monochromator provides a discrete narrow wavelength band from a selected part of the electromagnetic spectrum (ultraviolet, visible, infrared). The beam of light is focused on the photocell and the light intensity is measured. Light intensity is compared before and after a solution is placed in the beam of light. Spectrophotometry is useful in two ways. (1) It can be used to characterize and identify a com- pound. When the instrument is used for this purpose light absorption is measured over a range of wavelengths. Generally a graph, called an absorption spectrum, is made plotting the light absorption versus wavelength. (2) Spectrophoto- magnetic spectrum. The figure above shows metry can also be used to determine the concen- characteristic spectra for salmon sperm DNA and tration of a compound in solution. When the egg albumin, a protein. Notice first that while the instrument is used for this purpose the amount of heights of the two absorption maxima are the same, light absorbed by a solution at one wavelength is they occur at different wavelengths, 280 nm for the determined. Both of these uses of spectrophoto- protein and 260 nm for the DNA. Second, the metry will be demonstrated in this exercise. concentration of the two solutions is very different: 0.1% for protein and 0.007% for DNA. These differences result from the number and nature of the Spectrophotometry identifies compounds absorbing groups in the two compounds. DNA absorption at 260 nm is caused by the purine and Oxyhemoglobin and deoxyhemoglobin differ in pyrimidine bases in the molecule. Protein composition only by the presence of oxygen bound absorption at 280 nm is caused by the amino acids to the heme of oxyhemoglobin. Binding of oxygen tryptophan and tyrosine in the molecule. Purine and to the large hemoglobin moiety makes a profound pyrimidine bases are stronger absorbers per residue difference in the absorption spectrum of these than the amino acids. More bases are also found per molecules. The comparison in this laboratory of unit weight of nucleic acid than are the tryptophan these two “red” heme compounds with one another and tyrosine per unit weight of protein. As a result, points out how spectrophotometry can be used to nucleic acids have a higher absorption coefficient identify molecules. (optical density per unit concentration) than For further illustration and example consider proteins. most proteins and nucleic acids. In contrast to the 1 CELL BIOLOGY SPECTROPHOTOMETRY Measurements & Laws of Spectrophotometry Spectrophotometry determines concentration The amount of light absorbed may be expressed As a demonstration of the use of spectrophoto- in two different ways. One way is as the percent of metry to determine concentration, the concentration the total incident intensity that passes through the of a protein will be established. Because, as sample mentioned above, most proteins do not absorb in the visible spectrum and this laboratory uses only %T = I/I x 100% o visible light spectrophotometers we must use a where Io is the incident intensity and I, the intensity means to “color” the protein so it will absorb in the of light leaving the sample. The other way of visible spectrum. A simple protein assay will be expressing the amount of light absorbed is the used; the Biuret test is specific for the presence of optical density (O.D.) or absorbance (A) peptide bonds. Known concentrations of proteins will be used to construct a standard curve, which is A = log (Io/I) = – log T. a plot of absorbance versus concentration. Using If a parallel beam of monochromatic light passes this curve you will be able to obtain the through a homogeneous absorbing medium, the concentration of protein of unknown concentration. intensity the radiation is decreased by a constant fraction for each unit thickness of the medium. This relationship between amount of radiation absorbed PROCEDURES and thickness of the absorbing substance is known as Lambert’s Law. 1. Qualitative Absorption In solutions, the absorbance is proportional to the number of molecules of absorbing substance per Direct the hand spectroscope toward outdoor unit volume of solution. Beer’s Law states that light. Adjust it such that a complete spectrum can there is a linear relationship between concentration be observed. Then place colored filters in front of in moles per liter and absorbance, and only holds if the slit and observe what wavelengths are the specific absorption per molecule does not vary attenuated. with concentration. These two laws may be combined into the Beer-Lambert Law 2. Operation of the Digital “Spectronic 20D+” A = αcl 1. Plug in the spectrophotometer. where α is the molar absorption coefficient, c is the 2. Turn it on with left front knob (%T). concentration of solute in moles per liter, and l is 3. Set wavelength with the knob on top. the path length in centimeters. Most spectrophoto- 4. Push the Filter selector lever (bottom, front) to meters have a linear % T scale; however, the correspond to the appropriate wavelength. convenience of this scale is not the most important 5. When the machine has warmed up (5 minutes) consideration. The last formula indicates that check to see that the “Transmittance” MODE is absorbance is linearly related to concentration. set. Therefore, absorbance is the most relevant scale for 6. Adjust the Transmittance to 0.0 using the LEFT most spectrophotometer applications. knob (%T) on the front of the machine. 7. Press the MODE button to read “Absorbance”. 8. Insert blank into sample holder, close the lid. 9. Adjust the Absorbance to 0.00 with RIGHT front knob (100%T/0A). 2 CELL BIOLOGY SPECTROPHOTOMETRY 10. Without changing any settings, remove the of the unknown concentration will be treated with blank, insert the sample and close the lid. Biuret reagent. The absorbance of all the samples 11. Read the absorbance. will be determined. The absorbance of the known 12. You may continue to place other samples in the concentrations will then be used to construct a machine and read their absorbance, or if you standard curve that will be used to determine the change the wavelength, repeat steps 8 through concentration of the unknown. 11. Note that all pipettings should be accurately done. Use one 2-ml pipette for the known concentration and its dilutions and a second 2-ml 3. Quantitative Absorption Spectra pipette for the unknown concentration. Prepare and examine hemoglobin and erythrosin 1. Place seven cuvettes in a rack. Label one “B” solutions. for blank and the remainder label 1-6. Pipette 2 1. Place about 10 ml of distilled water in a clean ml of distilled water into “B” and cuvettes 2-5. test tube. Using a Pasteur pipette, add about 2 Note do not add water to cuvette 1. drops of sheep blood. Cover the tube with 2. Pipette 2 ml of the 2.5 mg/ml stock albumin parafilm and mix by inverting several times. solution into cuvette 1. Pipette an additional 2 2. Divide this solution between two cuvettes. Set ml into cuvette 2. This produces a two-fold one aside; this is the “oxyhemoglobin”. dilution. 3. Add a small pinch of sodium hydrosulfite to the 3. Mix the protein and water in cuvette 2 by other tube. Cover this tube with parafilm again drawing the solution into the pipette and gently and invert several times. This is the blowing it back into the cuvette several times. “deoxyhemoglobin”. The color should be 4. Next withdraw 2 ml from cuvette 2 and add that noticeably bluer than in the other tube. to cuvette 3. Mix as before and transfer 2 ml 4. The oxyhemoglobin and deoxyhemoglobin from cuvette 3 to cuvette 4. samples are now ready to run. 5. Mix again and transfer 2 ml to cuvette 5. When 5. Fill two additional cuvettes about one third full cuvette 5 has been mixed, remove 2 ml and with the erythrosin solutions. One cuvette discard. Each cuvette should now contain should have the 1/20,000 concentration and the exactly 2 ml. Note also that each cuvette now second should have the 1/60,000 concentration. contains half the protein of the preceding 6. Use a fifth cuvette as a blank. cuvette in the series. 7. Using a Spec. 20, measure the absorbance in 10 6. Pipette 2 ml of the “unknown” protein nm increments of oxyhemoglobin, deoxyhemo- concentration into cuvette 6. globin, and the 1/20,000 and 1/60,000 erythrosin 7. To each cuvette now add 3 ml Biuret reagent solutions. Cover the range from 400 to 650 nm. and mix. Incubate at 37°C for 30 minutes. Think about how you want to do this with the 8. Using the blank first, read the absorbance of all fact that the “Spec” has to be “blanked” after the protein cuvettes at 550 nm in the Spec. 20. each change in wavelength. 4. Quantitative Determination of Protein Determine the concentration of a protein sample. A stock protein solution will be serially diluted by factors of two. These solutions along with a sample 3 CELL BIOLOGY SPECTROPHOTOMETRY DATA ANALYSIS Qualitative Absorption Compare your observations of the spectrum with the different filters. What region(s) of the spectrum would you expect a blue solution to absorb? A green solution? Can you make a generalized statement about “color” and absorbed wavelengths? Quantitative Absorption Plot all data for oxyhemoglobin, deoxyhemo- globin, and the two erythrosin concentrations on this graph.
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