10/5/06 Lab 3. Protein Determination Lab 3. PROTEIN DETERMINATION I. INTRODUCTION Reading in Biology, 6 th ed. By Campbell: PROTEINS-MANY STRUCTURES, MANY FUNCTIONS p. 71-80 Blood is a connective tissue with cells suspended in plasma p. 882-884. Protein is a major and indispensable class of cellular macromolecules. Accordingly, measuring protein concentrations in an amazingly diverse array of experimental contexts (How many grams of protein are there in a 27 gram serving of Fruit Loops?) continually engages the attention, and taxes the resources, of many investigators. The two essential and frequently cited basic references on methods for general protein determination are: Layne, (1957) and Peterson, (1983). These articles are on hard copy reserve for the class at the Science Library in the folder for this exercise. Two things impressed me as I read these papers. First, the methods described are all optical methods. That is, measurement of protein concentration relies on optical properties (absorption or turbidity) of protein solutions and these optical properties are measured with a device called a spectrophotometer (or colorimeter). Other types of methods (gravimetric, volumetric, etc.) for protein determination exist, such as the classic chemical method of Kjeldahl, but other such methods were not even discussed in these papers. Optical methods of biochemical analysis are convenient, sensitive and versatile. We will use optical methods for several additional exercises later in this course. Second, these papers described no fewer than 11 different methods for protein determination, even though they limited themselves to a presentation of optical methods rather than chemical methods. The existence of so many different methods must mean, I'm very, very sorry to say, that no single method is appropriate for all experimental needs in all laboratory contexts. Thus, before tackling the job of conducting protein assays an investigator is faced with the rather more challenging prospect of choosing the most appropriate method for a specific application. In this exercise you perform 2 different protein assays (the Biuret Assay and the UV absorbance assay) which, if nothing else, should demonstrate the versatility of optical assays. There will also be food for thought regarding the issues that you must consider when selecting an assay method for a specific application. 3.1
Lab 3. Protein Determination 10/5/06 You will use these assays to determine the protein concentration in an artificial unknown and in serum. 3.2
. 10/5/06 Lab 3. Protein Determination I.A. General Features of Biochemical Assays I.A.1. Dynamic Response Range and Sensitivity All assay methods have a characteristic dynamic response range. This is the range of sample concentrations over which the assay produces a differential response. Consider the situation shown in the graph below: 100% A B assay response 0% amount of protein in sample dynresp Assay method B exhibits a differential response over a wider range of values. However, method A responds to lower protein concentrations; concentrations that could not be measured by method B. We say that method A is more sensitive than method B. Therefore, method A may be the method of choice when samples contain very little protein. It is relatively easy to bring samples with too much protein into the response range simply by diluting them. Conversely, concentrating the protein in very dilute samples would usually not be practical. Therefore, in choosing an assay you want to use a method sensitive enough to detect the protein in your most dilute sample. I.A.2. Specificity A specific assay gives a response to the substance being assayed and to no other substance. Any other substance producing the same response as the substance being assayed is called an "interfering substance". Very few assays are 100% specific; virtually all respond to some interfering substance. The adept investigator will know what substances interfere with their assay and what interfering substances are possibly present in their samples. This information is sometimes available in the literature but it must often be determined by the investigator. Think about possible interfering substances in the 3 assays presented here. 3.3
Lab 3. Protein Determination 10/5/06 I.A.3. Sample Recovery In some situations the amount of sample available may be very limited. In such cases one would hope to recover the protein from the assay so that it could be manipulated subsequently. In this case one would want a "non-destructive" assay. Which of the 3 assay methods presented here is non-destructive? I.A.4. Logistics and Economics Under the heading of logistics we include a variety of issues related to the cost of materials and labor. You must consider the cost of equipment, chemical reagents, and staff time to prepare materials and perform assays. Increasingly, the cost of disposal of hazardous materials is becoming an important issue. I can assure you that these issues were carefully considered and are a significant constraint to the design of this laboratory exercise. I.A.5. Safety Prudent investigators will choose to avoid, whenever possible, methods that present risks to laboratory personnel or to the environment. See Section II. for a description of the hazards involved in this exercise. 3.4
10/5/06 Lab 3. Protein Determination I.B. Biuret Assay The Biuret reaction involves a reagent containing copper (cupric) ions in alkaline solution. Molecules containing 2 or more peptide bonds associate with the cupric ions to form a coordination complex that imparts a purple color to the solution with Amax = 540 nm. The purple color of the complex can be measured independently of the blue color of the reagent itself with a spectrophotometer or colorimeter. The Tetravalent Coordination Complex Formed by Proteins in the Presence of Biuret Reagent Protein assays based on the Biuret reaction were developed and used by numerous investigators throughout the first half of this Century. Typically each investigator adapted the basic reaction to suit their own needs, introducing their own variations of concentrations and assay conditions. Thus there are many versions of Biuret assay in the earlier literature. The Biuret Assay conditions normally specified by contemporary sources are based directly on the work of Gornall et al. (1948), usually without attribution. These workers conducted an extensive investigation of the effect of reagent concentrations, ph and incubation conditions to arrive at the recipes and procedures we now employ without much question. (Likely few people performing the Biuret Assay according to the procedure of Gornall et al. stop to ask themselves why the reagent contains potassium tartrate.) We do well to remember that these investigators were studying hepatic lesions, and therefore specifically optimized the assay conditions for determining proteins in serum. 3.5
Lab 3. Protein Determination 10/5/06 Using the Biuret Assay to determine protein concentration in an unknown sample requires, as a preliminary, construction of a standard curve made by assaying a series of protein solutions with known concentration. The protein bovine serum albumin (BSA) is frequently used for constructing standard curves because of its availability and low price. The typical Biuret procedure (as implemented in our procedure) is: Combine 0.9 ml Biuret Reagent with 0.3 ml of sample in a microcentrifuge tube and mix thoroughly by vortexing. Wait 5 minutes. Determine the Absorbance at 540 nm vs. a blank containing Biuret Reagent and di water, but no protein. I.C. UV absorbance Virtually all proteins exhibit a strong UV absorbance maximum near 280 nm. This characteristic absorbance is due almost entirely to the absorbance by the aromatic rings in the R-groups of the amino acids tryptophan and tyrosine. (As you will recall, these are 2 of the 20 amino acids ordinarily found in proteins.) Samples are assayed directly in a spectrophotometer capable of generating and measuring UV light at this wavelength. No chemical reagent is involved here, as in the Biuret assay, because the UV absorbance is an intrinsic property of the protein itself. 3.6
10/5/06 Lab 3. Protein Determination II. PROCEDURES We provide a standard stock solution of protein at [20 mg/ml]. The protein is bovine serum albumin (BSA). The first task will be to dilute the stock protein solution to give a series of solutions with different, known concentrations (Procedure II.A). This must be done first. The protein standard solutions will be used to construct standard curves for the 2 assay procedures (II.B, II.C). These can be completed in any order, though the availability of UV spectrophotometers is limited. Therefore, some groups should do the UV absorbance assay first. In addition to the protein standard solutions prepared in II.A, there will also be a simple "unknown" solution. The unknown is a pure solution BSA in water. Dilutions of the unknown should be prepared at dilution factors of 10-1 and of 10-2. This is summarized as follows: Finally, you will determine the protein concentration in a real sample of serum. Each pair of students will work with a reagent "Kit" containing the following: Biuret Reagent Bovine serum Albumin Protein Standard [20 mg/ml] Protein Unknown Serum Be sure you do not confuse the standard with the unknown protein solution. 3.7
Lab 3. Protein Determination 10/5/06 II.A. Protein Standards To construct reasonable standard curves for each of the 3 assay methods over a wide range (4 orders of magnitude!), you will need 5.0 ml of each standard solution. There are innumerable ways you might accomplish this. A suggested method is diagrammed below. 3.8
10/5/06 Lab 3. Protein Determination This scheme produces 15 samples (including the original stock) whose concentrations are known. They will be used to establish the relationship between protein concentration and absorbance (i.e. to construct a Standard Curve ). 1. Before coming to class, determine the protein concentration for each of the 15 tubes in the dilution diagram on the previous page. Record the concentration value in the diagram so that you can refer to it during lab. If you are not sure that your calculations are correct, you can check them on the web site. 2. Set up fourteen (14) 16 X 120 mm tubes in a rack. 3. Add the indicated volumes of di H2O to each tube. 4. Proceed with the dilution series by transferring the indicated volumes of protein solution. Be sure you MIX each tube thoroughly before making the next dilution. You now have 15 BSA standard solutions (the original stock we provided plus the 14 dilutions you just made) whose concentrations are known. 5. You can also make the 2 dilutions (10-1 and 10-2 ) of the unknown at this time. HAZARDS Biuret reagent is a strong base (ph >12) and toxic due to the copper. Whenever you work with Biuret Reagent solutions you must: WEAR GLOVES WEAR EYE PROTECTION CLEAN UP ANY SPILLS FOLLOW DIRECTIONS FOR PROPER DISPOSAL The protein standard solutions containing ONLY BSA are harmless. 3.9
Lab 3. Protein Determination 10/5/06 II.B. Biuret Assay Put on GLOVES and SAFETY GOGGLES 1. Add 0.9 ml Biuret Reagent to the appropriate number of microcentrifuge tubes. Note that there are 15 protein standards, including the 20 mg/ml stock (see diagram on p. 4.8). Use one tube per unknown. For example, if there are 3 unknown solutions and one blank (i.e. Biuret Reagent and water, no protein), you will need 15 + 3 + 1 = 19 microcentrifuge tubes. This Biuret Reagent can be done with the same pipette tip. Minimizing the # of tips you use saves money and the time it takes you to change tips. 2. Reset the volume of the P-1000 to 300 µl and transfer each of the protein solutions (or water, for the blank) to the tubes containing reagent. In this case the issue of whether or not you would need to change pipette tips for each transfer is more complicated than it was in step 1. If you begin with the most dilute sample, and work toward the most concentrated sample, then there should be no significant contamination of one assay tube by the other. However, there would be contamination of your standard dilutions with Biuret Reagent. Because you may need the remaining standard protein solution in the dilution tubes to repeat the experiment, we advise that you change tips for each transfer in this step of the procedure. 3. Cap and mix all tubes. Wait 5 minutes. 4. Look at the tubes carefully to see if the results are reasonable. 5. Determine and record the absorbance of each of the 15 standards and the unknown with your colorimeter. Are the values consistent with your visual inspection? Remember to use the Biuret Reagent + di water as a reference, not pure water. Be sure that the wavelength is set to 540 nm and that the proper filter is selected. It is probably best to simply decant (= pour) the solutions from the assay tubes to the cuvette. After each reading, return each sample to the microfuge tube it came from and save all the tubes until you are certain the experiment is complete and that the results are correct. HINT: If you begin with the most dilute sample, and work toward the most concentrated sample, you will not need to rinse the cuvette between samples. 3.10
10/5/06 Lab 3. Protein Determination 6. Liquid waste disposal: When you are satisfied that your results are correct, discard all the assay solutions (not the standards) by pouring the contents of each microcentrifuge tube into a waste beaker designated for Biuret Reagent. Rinse each tube with a small amount of di water from a squeeze bottle and add this rinse to the waste beaker. After rinsing, the empty microfuge tubes are placed in the receptacle provided. 3.11
Lab 3. Protein Determination 10/5/06 II.C. UV Absorbance In this assay we use the intrinsic ability of proteins to absorb ultraviolert (UV) light. The UV absorbtion of native proteins is due almost entirely to the presence of the aromatic amino acids tryptophan and tyrosine which have absorbtion maxima near 280 nm. The other 18 amino acids normally found in protein absorb weakly, if at all, in the UV range. For this assay you must use one of the Pharmacia/LKB Ultrospec spectrophotometers on the side bench. The colorimeters at your bench work only in the visible part of the spectrum; they have no source of UV light. Also, you must use the special UV-transparent cuvettes because the ordinary plastic cuvettes you use with your colorimeter are opaque to UV light. The UV-tranparent cuvettes are marked with small dots of red ink at the top. NOTE: You could use the UVtransparent cuvettes with your colorimeter at visible wavelengths, but you cannot use the ordinary cuvettes with the LKB instruments in the UV. There are rather lengthy printed instructions taped to the top of the LKB instruments, but your instructor may be able to give you a brief demonstration. The wavelength is set to 280 nm and the instrument is blanked using the "SET REF" button on a cuvette containing di H2O. (You need to blank the instrument only once.) You will probably want to use the P 1000 to transfer 1 ml from each standard tube to the cuvette. Again, work upwards, from lower protein concentration toward higher protein concentration so that you will not need to rinse the cuvette between samples. As you read each sample, return it to the tube in case you need to re-read a sample later. After you have read all the known protein standards, read the unknown and the dilutions of the unknown. 3.12
10/5/06 Lab 3. Protein Determination III. DATA ANALYSIS AND DISCUSSION Assemble your data for the known standard solutions in a spreadsheet. You will have separate columns for proteins concentration (mg/ml), Biuret absorbance values, and UV absorbance values. Simply ignore any absorbance values < zero. A. General Comparison of the Assay Methods The most straightforward way to compare the assay methods to each other (in terms of their sensitivity and response range) is to draw 2 standard curves on a the same log/log plot (log Absorbance values vs. log protein concentration). EXCEL makes this a snap because it eliminates to need to actually calculate logarithms. We urge you to take advantage of the opportunity to learn how to use EXCEL on the classroom computers. Using log scales rather than linear scales on the graph spreads out the data points that would otherwise be scrunched together at the lower end of the scales. Try it and see! Note that there is no log value for a negative numbers or for zero. You can simply eliminate them from the graph. (But you must note in your report that you have done this.) If you have many negative absorbance values it probably indicates that the sample concentration is below the detection limit of the method or that there was a problem with your blank. Also, dirty cuvettes, improper cuvette filling, and inserting cuvettes in the wrong orientation can cause this problem. The log/log plot should be labeled Fig. 1. Have your instructor check the plot. They will tell you whether you should use your own log/log plot for the discussion, or whether you should refer to the log/log plot provided on the web site. B. Determining Concentration of the Unknown Although the log/log plot can be enlightening with respect to the relative merits of the assay methods (you can immediately see the sensitivity and dynamic response ranges) the log scales tend to limit the precision with which you can determine the unknown concentration. To determine the unknown concentration more precisely you need a linear/linear plot of Absorbance vs.standard concentration that includes on the dynamic response range of the assay where Beer s Law applies. Plot linear/linear standard curves for the Biuret and UV assays sepatately as Fig. 2 and Fig. 3. Do not simply "connect the dots" in your graphs, but subjectively draw a best-fit straight line to the points. When drawing the line you may ignore any points that are obviously erroneous, but you must explicitly state that you have done so. When you have the standard curves properly plotted, perform the determinations of the unknown samples. 3.13
Lab 3. Protein Determination 10/5/06 Remember that your determined values are valid on when the absorbance value of an unknown (or its dilution) lies within the linear response range of the assay, i.e. that portion of the standard curve that follows Beer's law. You might need to use the absorbance value of the undiluted unknown, or the absorbance of one of the dilutions, depending on the assay and on the protein concentration. Expect to operate to some extent on a trial and error basis. Summarize the final results in a table in your notebook of the form: BIURET ASSAY Dilution used Absorbance Concentration (mg/ml) Corrected Concentration UV ASSAY Dilution used Absorbance Concentration (mg/ml) Corrected Concentration 3.14
10/5/06 Lab 3. Protein Determination IV. QUESTIONS AND DISCUSSION Which assay method shows the broadest response range and which assay method is most sensitive? Use the log/log plot (Fig. 1) to evaluate this. How might the results be affected if a different protein (other than BSA) were used to create the standard curve? Evaluate and discuss the pros and cons of each assay method. Do not confine yourselves to the features described in section I.A. Did the two assays give similar results for the protein concentration of the unknowns? If not, which assay do you think is most reliable, and why? The instructor can provide the actual value of the unknown. References on Reserve Gornall, A.G., Bardawill, C. J. and David, M. M. (1949) Determination of Serum Proteins by means of the Biuret Reaction. J. Biol. Chem. 177; 751. Layne, Ennis Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology, vol. 3, p. 447 (1957) Peterson, Gary L. Determination of Total Protein. Methods in Enzymology, vol. 91, p. 95 (1983) 3.15