BC2004, Spring Semester 2005, Exercise 1-1

BC2004, Spring 2005 Lab Exercise 1

Spectrophotometric Study of Biomolecules: Absorption Spectrum of Hemoglobin and Quantitative Assay of Serum Protein Concentration

Spectrophotometry is a technique frequently used in biological laboratories; it is based on the measurement of relative light intensities. In lab exercise 1, you will be introduced to the concepts and applications of spectrophotometry through the use of a spectrophotometer, which you will use in several other lab exercises this semester. Many kinds of substances absorb selective wavelengths of visible light (400 to 700 nm) and reflect and transmit other wavelengths. As a result, they appear colored. Substances that absorb visible light are called pigments. You will use spectrophotometry this week to study several colored compounds. Other substances do not absorb visible wavelengths of light and appear colorless. We can indirectly study such colorless compounds using spectrophotometry if they react with other substances to form colored products. We can also use a specialized spectrophotometer to measure light absorption in a region of the electromagnetic spectrum that is not visible to the human eye (e.g., DNA appears colorless, but absorbs certain wavelengths of ultraviolet light and can be studied using a UV-spectrophotometer). We will use both of these methods later this semester. Spectrophotometry measures the absorption of light of specific wavelengths and thereby provides two types of analysis: qualitative and quantitative. Qualitative analysis is employed to help identify a substance by determining the proportion of light across a spectrum of wavelengths that is absorbed by a solution of the substance. Quantitative analysis is employed to estimate the concentration of the substance in a solution. Over a certain range, the proportion of light absorbed is directly dependent on the concentration of the substance. To maximize the sensitivity of the measurement, we use the wavelength at which the substance absorbs the highest proportion of light (its absorption maximum). Spectrophotometer basics A spectrophotometer is the analytical instrument you will use today; it consists of two principal parts: a spectrometer and a photometer. Using a white (and/or UV) light source and a monochromator (a prism), the spectrometer of the instrument provides discrete wavelengths of light at a known intensity (incident light). The photometer consists of a photoelectric tube sensitive to the wavelengths of light provided by the spectrometer and a galvanometer to quantitate the intensity of transmitted light. The liquid sample to be measured is inserted between the spectrometer and the photometer. By comparing the light intensity emitted by the spectrometer to the light intensity measured by the photometer (that has been transmitted by the solution), the solution’s absorbance is calculated by the instrument:

A (absorbance) = log10 (intensity of incident light / intensity of transmitted light)

BC2004, Spring Semester 2005, Exercise 1-2

Figure 1. Simplified spectrophotometer diagram. A spectrophotometer directs light of a specific wavelength into the solution. This is the incident light. The light that passes through

the solution is the transmitted light; it is sensed by the photoelectric tube and quantified by the galvanometer. [A more detailed figure will be reviewed during recitation.] A. Absorption spectra and color Every compound has a unique absorption spectrum, a distinct set of wavelength(s) it absorbs. A solution of a particular compound, such as hemoglobin, always absorbs light of specific wavelengths and does not interact with light of other wavelengths. At any concentration (an absorption spectrum is independent of concentration), a solution of a light-absorbing compound will absorb a relatively high proportion of light of certain wavelengths, a moderate proportion of light of certain other wavelengths, and little or none of still other wavelengths. This pattern of peaks and valleys of light absorption is the compound’s absorption spectrum, a display of the relative proportion of light absorbed from each wavelength of the spectrum (see Figure 1 as an example). The wavelength(s) absorbed by a substance in the visible part of the spectrum are complementary to the color that we perceive. Color is a function of human perception, but absorption of specific wavelengths of light is a function of molecular interaction with light. If a substance absorbs blue and red light, but not green light, we will see it as green because that is the only light that reaches our eyes from that substance. For example, the absorption spectrum of a green substance should show low absorption of wavelengths in the green part of the spectrum, but may show high absorption of light in the red and blue parts of the visible spectrum. This is an absorption spectrum of a specific green pigment:

Figure 2. Absorption spectrum for a specific green pigment

0.000

0.050

0.100

0.150

0.200

0.250

0.300

400 500 600 700wavelength (nm)

abso

rban

ce

incident light of specific wavelength

some light is absorbed by the solution

transmitted light

BC2004, Spring Semester 2005, Exercise 1-3

What color is hemoglobin? Knowing just the answer to this question, what can you predict about the absorption spectrum of hemoglobin? While you cannot predict the details of its

absorption spectrum, you should be able to guess which wavelengths should be absorbed less and which may be absorbed more. In the first part today’s laboratory exercise, you will determine the absorption spectrum of hemoglobin. B. Calculating concentrations The basis of this application of spectrophotometry is that the proportion of light of a given wavelength absorbed by a solution of a particular compound is a function of the concentration of the solute. This allows quantitative analysis of the concentration of a substance using the Beer-Lambert relationship. From the absorbance value, the concentration of the solute can be calculated using the Beer-Lambert equation:

A = E x C x L where:

E (Molar Extinction Coefficient) = Absorbance of a l M solution of the substance measured through a l-cm light path. This is a constant for each substance at a specific wavelength.

C = molar concentration, in moles/liter. L = length of the light path through the solution, in cm. For the spectrophotometer you

will be using today, L is equal to 1 cm, so C = A / E.

In order to apply the Beer-Lambert equation, the Molar Extinction Coefficient (E) for the specific substance and wavelength must be given, measured, or calculated. If the substance does not absorb light strongly, its E value can be measured directly, as it is the absorbance of a 1 M solution (hence the name Molar Extinction Coefficient). However, for a highly colored substance, the absorbance of a l M solution is too great to read directly in a spectrophotometer and must be calculated from a standard curve. For most of the solutions you will use in the spectrophotometer this semester, the absorbance will be too great to read directly, so you will need to know how to make and use a standard curve. A standard curve is produced by measuring and plotting the absorbance of a series of dilute solutions, each of a known (i.e. standard) concentration. When Absorbance is plotted against concentration, the slope of the line is the relationship between concentration and absorbance. Remember that E is the absorbance of a 1M solution; it is also the slope of the standard curve. If it is too great to be measured directly, you can use the equation of the line on your standard curve to calculate E. Once E has been determined (either directly by measuring the absorbance of a 1M solution or indirectly through construction of a standard curve), the concentration of any solution of that compound can be determined by measuring its absorbance of light at the same wavelength.

(equation 1)

(equation 2)

BC2004, Spring Semester 2005, Exercise 1-4

Data, Standard Curve, and Calculation Example

Table 1. Standard Curve Raw Data Agent A concentration (M) absorbance at 400nm

0.1 0.07 0.2 0.14 0.3 0.21 0.4 0.27 0.5 0.39 0.6 0.42

Figure 3. Standard curve example.

y = 0.7314x - 0.006

0

0.1

0.2

0.3

0.4

0.5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

concentration (M)

abso

rban

ce a

t 400

nm

Excel was used to plot the data in Table 1 (giving Figure 3), determine the best-fit line, and calculate the equation for the line. [Note that every axis on every graph for this course must be FULLY labeled, including units where appropriate (absorbance does not have units).] If you were to measure the absorbance of a solution of Agent A of unknown concentration and find that it was 0.25, how could you calculate the concentration of that solution? There are two methods by which you can use the standard curve in Figure 3 to calculate the concentration (in M) of a solution of unknown concentration. One way to do the calculation would be to use the given equation for the line, where x = concentration (which you want to find) and y = absorbance, which you measured in the spectrophotometer. So here, 0.25 = 0.7314x – 0.0006. Using simple algebra, you can solve for x = 0.34 M. The relationship is the same when concentration is expressed in units other than molarity, such as mg/ml (just make sure the units in the standard curve match the units you need for the unknown). A second way to do the calculation is needed when you are not given the equation for the line. Remember from your high school geometry that the equation for a line is “y = mx + b,” where m is the slope and b = the y-intercept. You can come up with the equation for the line yourself (as you will on your exams in this course) or you can use Excel (or another computer program) to give you the equation (as you will when working with real data in lab this week and in the

BC2004, Spring Semester 2005, Exercise 1-5

coming weeks). The y-intercept should always be very close to 0. Why? (You should be able to answer this question after completing this week’s lab exercises.)

Important: the slope of the line = E. The slope of a line, m, equals the rise (change in y or ∆y, which in this case is ∆A) of the line divided by the run (change in x or ∆x, which in this case is ∆C). So, the slope of a standard curve is m = ∆Y/∆X or m = ∆A/∆C Begin by using a ruler to draw a straight line that best fits the points on your graph (yes, this is somewhat subjective). You can choose any two points on your line of best fit to calculate ∆x (∆A) and ∆y (∆C), as long as those two points are on the linear part of the graph (which may not be linear over the entire range of concentrations). As an example from Figure 3: Using point 1 (x = 0.1, y = 0.07) and point 4 ( x = 0.4, y = 0.27) ∆y = 0.27-0.02 = 0.2 ∆x = 0.4-0.1 = 0.3 so ∆y/ ∆x (or ∆A/ ∆C) = 0.2/0.3 = 0.67 = slope of the line in Figure 1 [Note that the slope is the approximately the same whether we use points 1 and 4, 2 and 5, 1 and 2, etc. It’s ok that the math won’t come out exactly the same when using real data and that your hand-calculated numbers won’t exactly match the numbers in Excel.] Once we have calculated the slope of the line, and thus E, we can use the equation C = A/E, to determine the concentration of an unknown solution. If the absorbance of the unknown is 0.25 (using the same example as above), and E = 0.67, it is easy to calculate C = 0.25/0.67 = 0.37 M. This is not exactly the same as the concentration calculated using the line’s equation from Excel, but it is close. We can only use these methods to estimate the concentration of an unknown, we cannot prove that the concentration is an exact number (remember, NEVER use the word “prove” in a scientific setting!!). Which of these two methods of calculating concentrations of unknown solutions do you think is most accurate? If you answered the Excel method, you are correct. The computer can do a better job of making a best-fit line than you can with your ruler. However, the by-hand method does a pretty good job of estimating the concentration of an unknown. You should learn and know how to use both methods for this course. Today’s laboratory investigations In these exercises, you will use spectrophotometry in two ways. (See Appendix 1 - 1 for instructions on using the spectrophotometer.)

First, you will determine the absorption spectrum of hemoglobin by measuring and plotting its absorbance of light of different wavelengths. Second, you will construct and use a standard curve of protein concentrations to estimate the concentration of protein in an unknown serum solution.

(equations 3 and 4)

BC2004, Spring Semester 2005, Exercise 1-6

Before coming to lab this week (and every week):

Lab Outline/Flow Chart Instructions, Tips, and Example You are required to write a brief outline of each lab exercises’ procedure that will be checked by your instructor at the beginning of lab each week. These outlines take the place of the quizzes in BIOL BC2003 (as for the quizzes, be sure to come on time; if you arrive late, you will not receive credit for your outline). The outlines serve two purposes: 1) to ensure that you have read the lab procedure and are prepared for lab; and 2) to serve as a checklist for you to follow as you perform the lab. Many of this semester’s lab exercises are fairly complicated, and it will sometimes be easier for you to carry around and refer to your outline than to your entire lab notebook. Your outline should be concise, but complete enough that your lab partner could complete the lab using only your outline and the lab handout. You may paraphrase or abbreviate what you see in the lab notebook, but not so much that you alter the procedure or cannot understand it. As you deem appropriate or as specified by your lab instructor, you may include specific amounts and times. For some labs, it may be more helpful to you for your outline to be in checklist form. For other weeks, the methods are more complicated and it may be more helpful to prepare a flow chart that shows the procedures using diagrams in additions to words. Use your judgment unless your lab instructor specifies one form or the other. Remember that it is never acceptable to use information from any source (including your lab handout) without citation. Try to paraphrase the instructions from the lab handout as much as possible; do not copy word-for-word unless it is absolutely necessary (and use quotation marks when you do). Please specifically refer to the lab handout as the source of your methods and cite it using the format in the sample below (also presented in A Short Guide to Writing About Biology). If you get information from a source other than the lab handout, please paraphrase that information and cite it according to the instructions found in A Short Guide to Writing About Biology.

BC2004, Spring Semester 2005, Exercise 1-7

BC 2004 Partial Sample Outline

Note: Outlines must be hand-written, but your handwriting doesn’t have to be this neat ☺. Lab 1: “Spectrophotometric Study of Proteins: Absorption Spectrum and Quantitative Assay of Proteins” (BC2004 Lab Exercise 1 Handout. January 2004. Pp. 1-13. Barnard College. New York, NY.) Purposes 1. To determine the absorption spectrum of hemoglobin (purified from ox blood) from 400-700 nm. 2. To determine the absorption of different, known concentrations of bovine serum albumin (BSA) reacted

with Biuret reagent to construct a standard curve showing the relationship between protein concentration (in mg/ml) and absorption at 550 nm (absorption peak for Biuret product).

3. To use the BSA standard curve to determine the protein concentration within calf serum. Procedure (Adapted from BC2004 Lab Exercise 1 Handout. January 2005. Pp. 1-1 – 1-20. Barnard College. New York, NY.)

Part A. Absorption Spectrum of Hemoglobin

1. Put piece of white paper in cuvette. Put cuvette in spectrophotometer. Leave door open on spectrophotometer and change wavelengths from 650nm to 400nm. Record the colors I see on Data Table 1.

2. Make predictions about absorption spectrum.

3. Prepare 2 cuvettes Reference/blank: Sample cuvette: 4 mL distilled water 4 mL diluted hemoglobin (purified from ox blood) 4. Blank spectrophotometer with cuvette 1 each time the wavelength is changed. Read absorbance of cuvette 2 every 10 nm from 400nm to 700nm. Record results in Data Table 1. You should complete this outline before coming to lab 1 (it will be worth 1 point today, but 5 points in future weeks). You will use your outline during lab. Remember that you might want to start with part B. You should clearly indicate the order you will do the exercises on your outline. Complete your outline on your own paper.

1 2

BC2004, Spring Semester 2005, Exercise 1-8

Methods Work in pairs today, but remember that your assignments must always be completed

individually (and in your own words). Note: you may want to begin with part B because it requires a 30-minute incubation. During that 30-minute incubation, you could work on part A. You should decide the order in which you would like to do the exercises before coming to lab, when you are preparing your flow chart. A. Absorption spectrum of hemoglobin Almost all of the oxygen carried in blood is combined with hemoglobin within the red blood cells. Hemoglobin is composed of a protein (globin) to which the chromophore, a red, iron-containing heme molecule, is attached. The absorption spectrum of hemoglobin in the visible range is similar to that of many other heme-containing proteins. 1. Place a strip of white paper in a cuvette and place the cuvette in the sample holder in the

spectrophotometer. Turn the light control knob all the way to the right. Leaving the cover open, rotate the wavelength control slowly from 650 nm to 400 nm. Record (in Data Table 1) the wavelengths at which the color of the incident light clearly changes color; you should see a changing spectrum from dark red light to pale violet.

2. Using the space available on page 1-12, record the color of your hemoglobin sample. Based on is colored appearance, predict which wavelengths it should absorb less and which it may absorb more. An important part of this exercise is learning how to accurately and precisely measure small volumes of liquid using pipettes. Please ask your lab instructor or teaching assistant if you have ANY questions about their use! 3. Label and prepare two cuvettes: Cuvette 1containing 4 ml distilled water Cuvette 2 containing 4 ml purified hemoglobin (from ox blood)

What was the concentration of hemoglobin you used? Write it down here. _____________________________________________________________ Cuvette l is the reference cuvette, or blank; cuvette 2 is the sample cuvette. When the reference cuvette is placed in the instrument, light absorption by the solvent (in this case, water) is measured. This is subtracted from the absorption by the solution, and the difference is accounted for by light absorption by solute only.

4. Follow the general instructions on how to use the spectrophotometer found in Appendix 1-1. Read the absorbance of the sample of hemoglobin every 10 nm from 400 to 700 nm.

Estimate the absorbance at each wavelength to the nearest hundredth (example 0.45). Record your data in Data Table 1 and plot the data using Excel (specific instructions will be provided later in this handout)

BC2004, Spring Semester 2005, Exercise 1-9

B. Biuret colorimetric assay of serum protein

Compounds with two or more peptide bonds (e.g., proteins) react with copper ions in an alkaline solution to form a violet-colored complex that absorbs light at 550 nm. This reaction is the basis of the Biuret assay for protein. Biuret reagent is a solution of CuSO4 in NaOH. The assay consists of two parts that will be conducted simultaneously. The first part consists of establishing a standard curve by using several known quantities of a pure protein. In this experiment, bovine serum albumin (BSA) will be used as the standard protein to be reacted with the Biuret reagent. Because essentially ALL proteins react with the Biuret agent in the same manner, this standard curve will give us a value for A per (mg protein/ml) that can be applied to any protein solution reacted with Biuret reagent. (Remember that the Beer-Lambert equation will also work with mg/mL units as well as with M units.) The second part of this assay consists of reacting measured quantities of serum (containing unknown amounts of protein) with the Biuret reagent and measuring the absorbance of each reaction mixture. Standard curve preparation 1. Prepare and label eleven test tubes as directed in the tables below. Use a pipet pump and a

one 1.0-mL pipet to dispense the amount of NaCl solution to all tubes as directed in the tables, a second 1.0-mL pipet to dispense the BSA solution, and a third 1.0-mL pipet to dispense the serum. Be careful not to contaminate your pipettes; if you do, please ask your instructor for clean pipettes.

Table 2. For the preparation of a standard curve with BSA

Tube

mL

NaCl

mL BSA (v1)

total mL per tube (v2)º

BSA concentration (c2) in

mg/mL*

1 (reference/ blank)

1.0

0

1.0

0

2

0.9

0.1

1.0

3

0.8

0.2

1.0

2 mg/ml (given as an example, you will

need to calculate for the other tubes) 4

0.6

0.4

1.0

5

0.4

0.6

1.0

6

0.2

0.8

1.0

7

0

1.0

1.0

º Do not add 1.0 mL of anything to each tube. This is simply the total volume in each tube. * Important: see calculation instructions on the following page! You can complete these

calculations before coming to lab if you would like to be more efficient during lab.

BC2004, Spring Semester 2005, Exercise 1-10

*You will need to calculate the BSA concentrations by using the equation c1v1 = c2v2. [c = concentration; v = volume.] The stock solution of BSA that you will use today is 10 mg/ml ( = c1, the starting concentration).

General instructions: c1v1 = c2v2

where c1 = starting concentration, v1 = starting volume, c2 = final concentration, and v2 = final total volume. For Table 3: c1 = 10.0 mg/mL (concentration of the stock solution), v1 = 0.2 mL (the amount of stock solution you used), c2 = concentration of BSA in the final tube (what you want to calculate), and v2 = 1.0 mL So, (10 mg/mL)(0.2mL) = c1 (1.0mL), and c1 = 2.0 mg/mL Table 3. For determining the concentration of serum protein

Tube

mL NaCl

mL serum

0.5X

total mL per tube

total dilution of serum **

8

0.9

0.1

1.0

0.05 X

9

0.8

0.2

1.0

10

0.7

0.3

1.0

11

0.6

0.4

1.0

**You will need to calculate the total dilution of serum in each tube. Keep in mind that the serum has already been diluted to 0.5X (meaning it is at half its original concentration, which was 1.0X) in water (a stock solution was made with 300mL serum and 300mL distilled water). You will need to understand these dilutions in order to calculate the concentration of serum proteins in each tube. Sample calculation: Tube 8: 0.1 mL 0.5X concentration + 0.9 mL NaCl = 1.0 mL total volume c1v1 = c2v2 c1 = 0.5X, v1 = 0.1 mL c2 = ?, v2 = 1.0 mL So, (0.5X) (0.1 mL) = c2 (1.0 mL), and c2 = 0.05X

(equation 5)

BC2004, Spring Semester 2005, Exercise 1-11

2. Once you have prepared the eleven tubes, mix the contents of each tube by covering the tube with Parafilm and inverting the tube several times. (Use a single strip

of Parafilm for all tubes, moving to a clean, unused area of the strip for each tube.) 3. Then, using a 5.0-mL pipet and a pipet pump (do not mouth-pipet Biuret reagent or

anything else, EVER, in ANY lab, at ANY time), add 4 ml of Biuret reagent to each tube and mix well by inverting, again using a clean, unused area of the Parafilm for each tube.

4. Allow the protein to react with the reagent for 30 min. 5. Read the absorbance of the solution within each tube at 550 nm and record the readings

in the data table. (It is usually not necessary to check the blank before each measurement, but the instrument may drift, so it is wise to check the blank occasionally and readjust it to read 100% Transmittance with the light control knob if necessary.)

WAIT—do not forget an important part of lab: Be sure to clean up after yourself in an appropriate manner before leaving the lab. Dispose of materials as specifically instructed and place all materials back where you found them. You are strongly encouraged (although not required) to stay in lab to work on your graphs and calculations with your lab partner.

BC2004, Spring Semester 2005, Exercise 1-12

Data Table 1. A. Absorption spectrum of hemoglobin

Color of hemoglobin ____________________________

Predict which wavelengths should it absorb less _______________________________________ Predict which wavelengths might it absorb more ______________________________________

Wavelength (nm)

Absorbance Absorbance (no units)

color you see*

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

560

570

580

590

600

610

620

630

640

650

660

670

680

690

700

*You do not need to fill in every box in this third column, but be sure the colors of the spectrum (ROYGBIV) and the ranges of their wavelengths are shown.

You will use this data to construct an absorption spectrum using Excel.

BC2004, Spring Semester 2005, Exercise 1-13

B. Biuret colorimetric assay of serum protein

Data Table 2. Standard Curve Data

Tube concentration of BSA (mg/mL) from Table 2 A550

1

2

3

4

5

6

7

You will use the data from Data Table 2 to construct two standard curves, one by hand and one in Excel. Data Table 3. Calculating the concentration of protein in diluted serum

Tube A550 calculated concentration of

diluted protein (mg/mL) using Excel line equation

calculated concentration of diluted protein (mg/mL) using C = A/E, where E = hand-calculated

slope of hand-drawn line)

8

9

10

11

Show your Data Table 3 calculations on the next page! Note that you cannot complete this table until after you have made your standard curves.

BC2004, Spring Semester 2005, Exercise 1-14

Room to show your work for the completion of Data Table 3:

Column 3: Equation from Excel line: ______________________ y = ________________ x = ________________ Tube 8: Tube 9 : Tube 10: Tube 11 : Column 4 of Data Table 3 : calculate the slope of your hand-drawn line = ∆y/∆x m = _ ∆y __________________ = ______________________ ∆x m = E = A = C/E, so C = __________ (basic algebra here) Tube 8: Tube 9: Tube 10: Tube 11:

BC2004, Spring Semester 2005, Exercise 1-15

Data analyses

You may work in pairs to complete only your Excel graphs and analyses during lab this week. Unless specified (as it is here), you should complete graphs and data analyses individually in future labs. All written parts of your reports should be completed individually. A. Absorption spectrum of hemoglobin 1. Open a new Excel file/workbook. On sheet 1 of your workbook, type the wavelengths for

which you measured absorbance in column A, beginning with the smallest wavelength at the top. In column B, type in the absorbance you measured at each wavelength.

2. Next, highlight your data in columns A and B. Go to the “Insert” menu, and choose “Chart.”

Choose an “XY scatter plot” from the left side of the dialog box that opens and the option on the top left of the right side of the dialog box (chart sub-type). Click on “Next” two times.

3. In the title dialog box that opens, choose an appropriate title for your chart and type it in the

appropriate box (something along the lines of “Figure 1. Absorption spectrum of hemoglobin purified from ox blood” would be fine, but do not use this exact title). For the value of the x-axis, type in “wavelength (nm)”. For the value of the y-axis, type in “absorbance” (note that absorbance does not have units).

4. Do not make changes in the “axes” dialog box. In the “gridlines” dialog box, you can turn

on/off gridlines as you please (whatever you think looks best is fine). In the “legend” dialog box, turn off the legend by deselecting the “show legend” box. Finally, make sure the “none” box is selected in the “data labels” dialog box. Click on “Next” again.

5. Place the chart as an object in “Sheet 1” (which is where your data are located). Click on

“Finish,” and your graph will appear on Sheet 1. You are likely to want to make some changes to your graph. In particular, change the values for the x- and y-axes so they are just a bit below and above your minima and maxima for your graph. To do this, double-click on an axis and change the maximum and minimum in the “scale” dialog box.

6. Print two copies of Sheet 1 (including your data and graph)-one for your lab report and one

for your lab partner’s lab report. 7. Write in your “color” values from Data Table 1 onto your graph (in some manner you believe

is appropriate). Data analyses (IN YOUR OWN WORDS, answer the following questions in your lab report): 1. What wavelength(s)/color(s) of light does hemoglobin absorb most? What color(s) does it

absorb least? How are these related to your predictions? How are they related to the color of hemoglobin perceived by your eyes?

2. If you wanted to find out the concentration of hemoglobin in a solution of unknown

concentration, which wavelength(s) would be good to use? Which would not be good to use? 3. If a solution of a substance displayed only one absorption peak, would it be safe to conclude

that it was chemically pure or to conclude that there was only one light-absorbing substance present? What might be a possible alternative conclusion?

BC2004, Spring Semester 2005, Exercise 1-16

B. Biuret colorimetric assay of serum protein By Hand—calculating protein concentrations of diluted serum solutions BSA standard curve. Plot BSA concentration in mg/ml on the x-axis and absorption at 550nm on the y-axis. Be sure to clearly label the axes and include units (as you should on every graph you make for this course). 1. On a page of graph paper, plot the absorbance readings of the BSA samples (on the Y-axis) as

a function of protein (i.e., BSA) concentration of the sample (on the X-axis). Label axes! 2. At least part of the data should generate a straight line; this is the region in which the

absorbance is a linear function of concentration. Using a ruler, draw as straight a line as possible (a “line of best fit”) through the points, including the origin (coordinates 0,0); why should your line go through the origin? (If the origin really does not fit in your line, do not include it.)

3. Using any two points on the straight line, calculate the slope of the standard curve. This is the

E value of the Beer-Lambert relationship between absorbance and concentration. You can calculate the slope from your graph: chose two points that fit well on the line: count up the

difference between those two points in the vertical direction (rise) and divide that by the difference between those two points in the horizontal direction (run). This is the change in y divided by the change in x. When I calculated this from my graph, there was a 0.06 difference in Absorption between my two points = difference in y. There was a 1.0 difference in BSA concentration between my two points = difference in x. So, the slope for my line = 0.06/1.0 = 0.06. Do not use this value for your calculations, but calculate your own slope from your own graph.

4. Determine the concentration of protein in each serum dilution whose A value is within the

linear region of the standard (BSA) curve; use the Beer-Lambert equation and the value for E that you have calculated from your standard curve. Complete the column 4 of Data Table 3 (there is room to show your work on page 1-14). Be sure to include units.

Using Excel—calculating protein concentrations of diluted serum solutions BSA standard curve. Plot BSA concentration in mg/ml on the x-axis and absorption at 550nm on the y-axis. Be sure to clearly label the axes and include units (as you should on every graph you make for this course). 1. Open a new sheet (sheet 2) in your Excel workbook. In column A, type in the BSA

concentration of your tubes for your standard curve. In column B, type in their absorbance at 550 nm. Following the instructions for graphing written in part A, chart your standard curve as an XY scatter plot. To add a trendline to your graph, go to the “Chart” pull-down menu at the top of the screen, and select “Add trendline,” type “linear”. In the “options” dialog box, select the “display equation on chart” box. This will give you the equation for your standard curve line. Use this equation to complete column 3 of Data Table 3 (there is room to show your work on page 1-14). Be sure to include units.

BC2004, Spring Semester 2005, Exercise 1-17

Calculating protein concentrations of the undiluted serum solution

1. Remember that you diluted the serum solution in tubes 8-11. You will need to calculate the concentration of protein in the original, undiluted serum solution.

How will you do these calculations? See Appendix 1-2 for some tips and examples. Each sample was a different dilution of the original serum solution, which was a 0.5X solution (or a 1:2 dilution) of calf serum. Calculate the concentration of the original serum sample as the mean of the values calculated from all suitable reaction mixtures. See Appendix 1 - 2 (on page 1-21) for more instructions on how to calculate the original concentration of protein in the undiluted calf serum. Complete the following table. Table 4. Final calculations of protein concentration in undiluted calf serum.

Tube

total dilution

of serum

concentration of protein in diluted solution

(mg/mL) using Excel

concentration of protein in UNdiluted calf serum (mg/mL)

(from Excel calculations)

8

0.05 X

9

10

11

Tube

total dilution

of serum

concentration of protein in diluted solution

(mg/mL) (using hand-drawn graph)

concentration of protein in UNdiluted calf serum (mg/mL)

(from hand-drawn graph)

8

0.05 X

9

10

11

Average concentration of protein in undiluted calf serum (from all 8 calculations if they are close, from a subset if you have reason to use only a subset): _______________________

BC2004, Spring Semester 2005, Exercise 1-18

Data analyses (IN YOUR OWN WORDS, answer the following questions in your lab report):

1. Why did you read absorbance at 550nm for your BSA standard curve and for your serum

solutions? Why not read absorbance at 400nm or 700 nm instead? (Check the absorption spectrum for BSA posted in your lab room for some clues.)

2. Why should the y-intercept (b in the line equation “y = mx + b”) ideally be zero (or very close

to zero) for a standard curve line? (Hint, think about what the ordinates (x = 0, y = 0) mean.) 3. Did your hand-calculations and computer-calculations give you comparable results of protein

concentration in calf serum? Which method do you think is likely to give more accurate results and why?

Writing your lab report (to be submitted at the beginning of lab next week, 1/31-2/4/05): This first lab report is not a formal lab report. However, your report should include the following: 1. Descriptive report title (pp. 206-208 in 5th edition of A Short Guide to Writing About

Biology). Under no circumstances should you copy the title of this lab handout. 2. A one- (or few-) sentence statement of the purpose of the experimental approach. 3. Your outline/flowchart/checklist of what you were to do in lab.

4. A summary of your hypotheses, predictions, and observations (e.g., sketches, numerical data,

calculations)—presented in an organized manner! 5. Conclusions and discussion, including answers to the questions in the laboratory handout. While there is not a strict format for this first lab report, please strive to make your report well organized, clear, and straightforward. I recommend breaking parts 4 and 5 of your report down by section. For example, you should begin with part A. Present your predictions, data, and discussion for this exercise together (in paragraph form), then move on to part B. Generally, it is easier to write a formal lab report that strictly follows the format presented in A Short Guide to Writing About Biology. However, in this case, you have not really performed an experiment. You didn’t have formal hypotheses, experimental design, and data analyses. This was more of an exercise to familiarize you with the use of pipetter and the spectrophotometer. While you did perform real calculations and determine the concentration of protein in a solution, you can’t come to the same kinds of conclusions about data from an experiment supporting or not supporting your hypotheses.

BC2004, Spring Semester 2005, Exercise 1-19

Appendix 1- 1 Use of the Spectronic 20 Spectrophotometer

Figure 4. Diagram of Spec 20.

A. Anatomy and functions This type of spectrophotometer consists of five external parts with which you should become familiar in

order to use the instrument.

1. The power knob (at the left on the front of the instrument); it is also used as the "zero control” or “dark control."

2. The “absorbance/transmittance control” or "light control" knob (at the right on the front of the

instrument); this knob is rotated to set the reading (see 5) to Absorbance = zero (which is equal to 100 % transmittance) when a reference/blank solution is in the light path.

3. The “sample compartment” (at the left on the horizontal table of the instrument); a cuvette is a glass

tube of known diameter that will be used to insert samples into the instrument; there is a cover on the cuvette holder that MUST be closed during all readings and adjustments of the instrument to prevent entry of light from the room into the photometer.

4. The “wavelength selector” knob and its scale (at the right on the horizontal table of the instrument);

rotating this knob rotates the prism of the spectrometer so that light of the wavelength indicated on the scale passes through the cuvette.

5. The “absorbance/transmittance display” meter (on the upper face of the instrument); the upper scale

displays % Transmittance, and the lower scale displays Absorbance; the light control knob (see 2) controls the movement of the needle across the meter when a cuvette is in place.

BC2004, Spring Semester 2005, Exercise 1-20

B. Operation

1. Turn on the Spectronic 20 and allow at least 15 min. for the instrument (and its light source) to warm up (and emit a constant light supply).

2. Set the dark/zero control so that the needle reads 0 % transmittance. 3. Set the wavelength with the wavelength selector knob. 4. Prepare a cuvette containing a liquid that contains all the components of the sample solution except

the substance whose absorbance is to be measured. This is the reference cuvette or blank. Wipe the outside of the cuvette with a KimWipe (to remove any drips or fingerprints). Place the reference cuvette in the sample holder, close the cover, and use the light control to adjust the needle to read 100 % transmittance. This is to make sure any absorbance you measure is from your compound of interest, not from the solution in which it is dissolved.

5. Replace the reference cuvette with a sample cuvette containing the sample solution, and close the

cover. Allow the needle to come to rest and read the absorbance (make sure the needle and its mirror image behind are lined up). (Note that the absorbance scale reads from right to left, whereas transmittance reads from left to right.)

6. To read another sample at the same wavelength, replace the first sample cuvette with the next one. 7. To read the same sample at another wavelength, remove any sample from the sample compartment,

set the new wavelength with the wavelength selector knob, insert the reference cuvette, adjust to 100% transmittance with the light control knob, then insert the sample, and read its absorbance.

Important: The instrument must be “blanked” or reset with the reference cuvette for each

any every wavelength you use.

BC2004, Spring Semester 2005, Exercise 1-21

Appendix 1- 2 Calculating concentrations and Dilution Factors

The concentration of a solute can sometimes be determined by direct measurement of the amount of material per unit volume of the solution. Very often, however, the actual concentration is too high to be measured accurately. In such cases, the measurement is made at a concentration suitable for the method of measurement, and the actual concentration is calculated from that value and the dilution factor. The calculation of concentrations is based on the premise that the total amount of solute does not change when the mixture is diluted; it is merely dispersed into a larger volume. This is expressed by the statement: v1 volume units of concentration c1 contains the same amount of solute as v2 volume units of concentration c2. We will always have a starting volume and concentration (designated with a subscript-1) and a final volume and concentration (designated with a subscript-2). The relationship between starting and final concentrations and volumes can be expressed through the equation:

c1v1 = c2v2

where c1 = starting concentration, v1 = starting volume, c2 = final concentration, and v2 = final total volume.

Concentrations can use any units, as long as they are the same units for c1 and c2. Simple algebra allows calculation of any one of the values c1, v1, c2, or v2 when the other three values are known. Here are four examples of uses of this relationship. Example # 1: The unknown is c2. In this exercise, you were provided with BSA at a concentration of 10 mg/ml. If you had mixed 5 ml of that solution with 10 ml of saline, what would the concentration of BSA have been in the diluted solution? c1v1 = c2v2 c1 = 10 mg/ml v1 = 5 ml (10 mg/mL) x (5 mL) = (c2) x (15 mL) c2 = what we want to find v2 = 5 + 10 = 15 ml c2 = (10 mg/mL x 5 mL) / 15 ml = 3.3 mg/ml The concentration of BSA would decrease in proportion to the ratio of volumes v1 and v2 because the same number of molecules of BSA originally present in 5 ml was dispersed into 15 ml. The ratio of v1 to v2 (5 ml/15 ml, or 5:15 = 1:3) is unitless and is known as the dilution factor. The same change in BSA concentration would have been achieved with any pair of volumes in that ratio: 5 liters diluted to 15 liters, or 1 microliter diluted to 3 microliters, or 25 pints diluted to 75 pints. Note that the dilution factor is unitless ONLY IF the two volumes are expressed in the same volume units.

(equation 5)

BC2004, Spring Semester 2005, Exercise 1-22

Example # 2: The unknown is c1.

An assay of dissolved sugar disclosed a concentration of 1.2 mg sugar/ml in a 1:50 dilution (meaning one part of the original, undiluted sugar solution was added to 49 parts water to give a final volume of 50 parts). Calculate the sugar concentration of the undiluted solution.

c1 = what we want to find c1v1 = c2v2 v1 = 1 part (c1) x (1 mL) = (1.2 mg/mL) x (50 mL) c2 = 1.2 mg/mL v2 = 50 parts c1 = (50 mL x 1.2 mg/mL)/1mL = 60 mg/mL Example #3: The unknown is v2. You have 0.5 liter of salt solution containing 5% NaCl. How much artificial seawater (3 % NaCl) can you prepare for your pet squid? c1 = 5% c1v1 = c2v2 v1 = 0.5 L (5 %) x (0.5 L) = (3 %) x (v2) c2 = 3 % v2 = what we want to find v2 = (5 % x 0.5 L)/ 3 % = 0.83 L Example #4: The unknown is v1. You try one now: You accidentally spilled a flask during lab. Your lab instructor is nice about it, but insists that you make more of the solution for your labmates to use. The flask contained 500 mL of 400 mg/mL hemoglobin. You have water and a stock solution of 4g/mL hemoglobin at your disposal. Ok, this one is a bit trickier, because the units for c1 and c2 must match (as well as the units for v1 and v2. Here, the units for c1 and c2 do not match and must be converted before you can use the c1v1 = c2v2 equation. Conversion first: 400 mg/mL hemoglobin = 0.4 g/mL hemoglobin Calculations second: c1v1 = c2v2 space to complete your calculations: c1 = 4 g/mL v1 = what you want to calculate c2 = 0.4 g/mL v2 = 500 mL Give your recipe for making the solution: _________mL of stock solution (4 g/mL) + ___________mL of water to make a final volume of _______________mL of (0.4 g/mL) solution.

BC2004, Spring Semester 2005, Exercise 1-23

Example # 5: Correcting for dilutions. CORRECTED

You calculated the concentrations of a series of diluted solutions of Agent C12 to be the following:

assay tube

mL water

mL of 0.5X Agent C12

total volume per tube

total dilution of Agent C12 in tube*

concentration of Agent C12 (mg/mL)**

1 9 1 10 0.05 X 25 2 8 2 10 0.1 X 50 3 7 3 10 0.15 X 75 4 6 4 10 0.2 X 100

* Calculated using the instructions on page 1-10. ** Calculated using equation of line for standard curve. To find the concentration of Agent C12 in the original, undiluted solution:

Tube 1: (25 mg/mL)/ 0.05 = 500 mg/mL undiluted concentration Tube 2: (50 mg/mL)/0.1 = 500 mg/mL undiluted concentration Tube 3: (75 mg/mL)/0.15 = 500 mg/mL undiluted concentration Tube 4: (100 mg/mL)/0.2 = 500 mg/mL undiluted concentration

Should we be concerned that these values all came out to be the same? Should your values for protein concentration in undiluted calf serum all come about to be approximately the same? Why or why not?

Materials

For the class: 4 Spectronic 20 spectrophotometers. Graph paper Parafilm strips (approximately 2 x 6 inches) For each pair of students: 13 Spectronic 20 cuvettes 3 1-mL pipettes with pipet pump 3 5-mlL pipet with pipet pump 5 mL purified hemoglobin in distilled water, 100 mg/mL 4 mL BSA (Bovine Serum Albumin) in distilled water, 10 mg/mL 2 mL calf serum diluted 1:2 5 mL distilled water 10 mL 0.9 % NaCl 50 mL Biuret reagent