THE STUDY OF THE OXYGENATION OF FERRO-HEMOGLOBIN



THE STUDY OF THE OXYGENATION OF FERRO-HEMOGLOBIN

GROUP R5

Vu Hong

Brett Riccio

Rebecca Lai

Chris Lindsey

Natalie Georgakis

Department of Bioengineering

University of Pennsylvania

April 30th, 1997

Abstract

Our objective in this experiment was to gain an understanding of the oxygenation of ferrohemoglobin. Hemoglobin is an important protein in the blood whose heme group enables it to carry oxygen to the cells in the body and take away their waste products. By studying how oxygenation relates to a peak weak wavelength through spectrophotometry, a method can be devised to detect the amount of oxygen in a patient’s sample of blood. In specific, we investigated three things about oxyhemoglobin and deoxyhemoglobin: their peak wavelengths, the molar coefficients of each at their peak, and the relationship between their peak wavelengths and different concentrations of oxyhemoglobin. To achieve our goals, we analyzed three different samples of equine blood, performing three trials on each. To extract the hemoglobin , we used an IEC PR7000 Centrifuge to spin down the red blood cells and then lysed them with deionized water. Each sample was diluted so that its absorbance could be read on the Milton Roy Spectronic 20 Spectrophotometer for a range of wavelengths from 350-600nm. Then, we used the Beer Lambert Law to analyze our results. We found the peak wavelengths to be 410nm for oxyhemoglobin and 421nm for deoxyhemoglobin. In comparing these values to the literature values of 412nm and 420nm, our error was 0.485% and 2.093%, respectively. Secondly, we graphed our absorbance values versus concentration to find our molar coefficients to be 15.9*104 (0.082 L/mol-cm and 15.1*104 (0.151L/mol-cm. We had to compare these values with the coefficients at the literature peak wavelengths, 12.4*104 for oxyhemoglobin and 11.5*104 for deoxyhemoglibin, and found our error to be 28.47% and 31.30%, respectively. For the last objective, we found a linear relationship of an R squared value of 0.9426 between concentration of oxyhemoglobin and peak wavelength.

Introduction

The study of blood is one of the most important and ongoing research areas in medicine. Blood transports nutrients to all the cells in our bodies. It fights sickness and disease by channeling white blood cells and platelets to cites of infection. It also serves as a “shuttle service” for the oxygen that cells need, and the carbon dioxide that they must excrete. Since blood has such a multifaceted role in the life functions of almost every living organism, it is essential to medical science that the processes and mechanisms of the blood are known in great detail. For our project, we studied the oxygen transport mechanisms of the blood. Blood uses a heme, or iron group, called hemoglobin to transport oxygen to every cell in the body. Hemoglobin has a strong affinity for oxygen, and when deoxygenated blood passes through the alveoli in the lungs, oxygen attaches to the sixth coordination position on the hemoglobin. Then, it is carried away through the entire body by way of arteries, arterioles, and capillaries. Along this path, cells absorb oxygen in order to complete chemical reactions essential to their individual function.

The properties of hemoglobin are extremely important because they directly affect both the quantity and rate at which cells in the body receive oxygen. There are many adverse effects of inadequate oxygen supply. Due to the high oxygen requirements of sensitive organs such as the brain, decreased supply can cause grave damage in a matter of minutes. In situations where the oxygen content of the blood is questioned, there must be a test to determine the level of oxygenation in the blood.

One powerful technique of measuring the oxygen content in the blood is absorption spectrophotometry. In visible spectrophotometry, the type we utilized, a beam of monochromatic light is focused through a sample of aqueous hemoglobin. The wavelength of this light can be controlled for each experiment. As the light passes through the sample, molecules that have energy levels corresponding to the wavelength will absorb light energy and become energized. This absorbance is then measured as the decrease in energy between emitted light and the resultant light.(BE 210 lab manual) These results can be analyzed with use of the Beer Lambert Law which states:

A=((()Cl

where the absorbance (A) is equal to the product of the molar extinction coefficient as a function of the wavelength, times the concentration of the sample times thickness of the material (l). The molar extinction coefficient is defined as the sum of the scattering coefficient and the molar absorption coefficient.(Castellan, p. 586) Values for this coefficient can be found in sources such as CRC Handbook of Biochemistry and Molecular Biology.

In order to use this method, hemoglobin is extracted from the blood through a process of centrifuging and washing the sample of blood (see Methods section). The hemoglobin is then run through a spectrophotometer at the peak absorbance wavelength for oxy and deoxy hemoglobin, 410nm, and 421nm respectively, to determine the amount of oxygen present. Our project deals with the process and experimentation required to develop a curve that will allow easy assessment of the amount of oxygen in a sample of blood from the results of such a “spec” test.

Background

Hemoglobin is an oxygen carrying molecule found in red blood cells. It is made up of 4 globin chains (2 ( chains, and 2 ( chains). Each of these chains contains a heme group of either ferrous (Fe2+) or ferric (Fe3+) iron, which is anchored to the chain by four nitrogen atoms. The ability of hemoglobin to attach oxygen is altered depending on which type of iron is attached. Ferrohemoglobin is created through the bonding of ferrous iron, and ferrihemoglobin is created through the attachment of ferric iron. Only ferrohemoglobin can attach oxygen, so we will only concern ourselves with this type of hemoglobin. The iron group can form two additional bonds, one of these (fifth coordination position) is filled by a histidine residue F8, and the other (sixth coordination position) is occupied by oxygen.(Stryer, p.151)

Oxygen attaches cooperatively to hemoglobin, that is, when one molecule of oxygen attaches, the hemoglobin is “encouraged” to bond the other three sites to oxygen. (Stryer, p.150) This process occurs in the alveoli of the lungs, before the oxyhemoglobin is sent through the body. In the body, cells create CO2 and H+ in response to glycolysis and cellular respiration. When the oxyhemoglobin passes by these cells, the CO2 and H+ actually encourage the release of these bonded oxygen molecules, and subsequently attach to the hemoglobin for transportation back to the lungs. In the lungs, the reverse process occurs, where oxygen encourages the release of H+ and CO2. These two stages of hemoglobin are called oxyhemoglobin and deoxyhemoglobin (Stryer, p.151)

|Form |Oxidation State |5th Coordination Position |6th Coordination Position |

| |of Fe | | |

|Deoxyhemoglobin |+2 |Histidine F8 |Empty |

|Oxyhemoglobin |+2 |Histidine F8 |O2 |

(Stryer, Biochemistry p. 151)

We are developing a spectrum that will differentiate between oxyhemoglobin (sixth coordination position filled) and deoxyhemoglobin (sixth coordination position empty). The quaternary structure of hemoglobin changes with oxygenation. This happens when two of the four oxygen bonding sites rotate around the other pair at about 15 degrees. The terminal residues on oxyhemoglobin have rotational freedom, something that deoxyhemoglobin lacks. These things render oxyhemoglobin quite different from deoxyhemoglobin, and these differences not only increase function, but they allow for differentiation as well. Further, oxy- and deoxy- hemoglobin absorb different wavelengths along the visible spectrum of light. By isolating these different wavelengths, and setting up spectrums, we will be able to determine the oxygen percentage of an unknown sample of hemoglobin.

We hypothesize that as the hemoglobin increases or decreases in oxygenation, its peak wavelength will correspondingly increase or decrease between the two extremes. We will therefore develop a proportional relationship in which absorbance wavelength of a sample will place that sample in a relative position between fully oxygenated and fully deoxygenated hemoglobin. By use of the lever rule (Callister, p. 243) we will be able to determine approximate percentages of oxygenation in our sample. Due to the medical importance of these studies, there has been extensive research to determine the expected peak wavelengths of many types of hemoglobin. The expected peak wavelengths for both oxy and deoxy hemoglobin are represented below, along with the accepted values for the molar extinction coefficients. As we do not have access to human blood samples, we based our experiments on equine blood. Below a comparison is made as to the expected differences between human and horse hemoglobin. While the values of the peak wavelength and molar extinction coefficients are well known for human blood sample, they are extremely difficult to find for horse or other mammal hemoglobin. For our experiment we will assume that the coefficients remain consistent between samples of horse and human hemoglobin. Some literature values are listed below.

| |Human Hemoglobin |Human Hemoglobin |Horse Hemoglobin |Horse Hemoglobin |

| |wavelength (nm) |e (L/mol-cm) |wavelength (nm) |e (L/mol-cm) |

|Oxyhemoglobin |523 |0.43*10-4 |412 |12.4*10-4 |

|Deoxyhemoglobin |542 |1.57*10-4 |541 |1.52*10-4 |

(Merck Index)

Current methods of determining the percent oxygen saturation in blood involve use of a multi-wavelength co-oximeter that determines the amount of oxygen that is present in a given sample of blood through measurement of the absorbance through a range of wavelengths of the sample. Another device is the Hemox Analyzer, which determines the amount of oxygenation in the blood through measurement of the absorption of monochromatic light by the hemoglobin. Many of these devices are set up so that they are directly inserted into the veins, so that inaccuracies do not result during removal of the blood. These tests provide the standards to which we will measure our results.

Decreased concentration of oxygen in the blood is a very serious issue, but without an accurate method of testing, it is difficult to recognize. Symptoms include dizziness, fatigue, cold extremities, and disorientation. Continued or acute decrease in oxygen concentration can lead to hypoxemia. Emergency rooms must be able to determine the amount of oxygen in the blood, in order to determine the appropriate treatment. Carbon monoxide poisoning is the most frequent form of oxygen concentration decrease, as CO bonds with hemoglobin some 200 times more readily than does oxygen.(Stryer, p.152) If a patient is admitted with limited CO poisoning, a treatment of high-rate concentrated oxygen is usually sufficient. The CO begins to lose its ability to occupy the sixth site, and oxygen is soon able to bond with the heme group again. When people have a worse exposure, and the CO poisoning reaches higher levels, it is necessary to put the patient into a hyper-baric chamber of 3-5 atm, so that the CO is forced off the hemoglobin by the increased concentration of oxygen.(Dr. Kris Brickman MD.) These treatments have proven very effective, and the patient is usually free of CO in 20 minutes to an hour.

Our experiment provides a way to quickly determine the oxygen content of a sample of hemoglobin. We effectively reproduced the results that have been accepted as correct, and in doing so have gained a better understanding of the ways that these values are determined.

Methods and Materials

Materials

• Milton Roy Spectronic 20 Spectrophotometer

• International Equipment Company PR7000 Centrifuge

• 13 mm cuvettes for the spectrophotometer

• Equine(Horse) Blood

• Sodium Dithionite, Powder, purified

• Assorted beakers, flasks, and pipettes, including Pipette-Aid

• 50 mL test tubes

• Parafilm

• Labview

Methods

Extraction of Hemoglobin from Horse Blood

The horse blood used was taken from horse #17 named Rye Catcher through a vacuum into a 75mL bottle containing anticoagulant citrate dextrose solution. The bottle noted that the blood was expired on 10/15/96 indicating that the blood could no longer be used for transfusions. In order to prevent any denaturing of the blood by exposure to extreme conditions such as heat, the blood was refrigerated when not in use.

The extraction of hemoglobin from the horse blood was a relatively easy process, but it required an investment of time. The first step was to fill two seal-able plastic test tubes with horse blood. The tubes were sealed tightly and placed in the centrifuge. The first “spin” was done at 13(C, 3000 rpm, for 13 minutes. When the spin was completed, a mass of red blood cells should have settled at the bottom of the test tube. The top, liquid portion of the test tube contents was discarded, using caution not to lose any of the red blood cells in the process. The red blood cells were still suspended in a mass of plasma, and other blood constituents. The test tubes were filled with .09% saline solution, and the top sealed. The tubes were shaken vigorously so that the settled red blood cells became fully dissolved into the saline. This new substance was centrifuged at the same conditions (13( C, 3000 rpm, 13 min). Again, the red blood cells were found settled at the bottom of the test tube, but this time they were “more clean,” that is, they had less plasma and white blood cells mixed in with them. The liquid was poured off, again being careful not to lose any of the solid red blood cell mass. This “cleansing” was repeated by adding more saline solution and centrifuging under the same conditions. At this point there was a relatively solid and concentrated mass of red blood cells at the bottom of the test tube. After pouring off the liquid once more, we were left with a test tube of clean, red blood cells. To extract the hemoglobin from these red blood cells we had to lyse the cells, or break them open to expose their contents. This was done by the addition of de-ionized water to the mass of red blood cells. The test tube was sealed and shaken vigorously to ensure that the red blood cells were thoroughly dissolved in the water. A few minutes were allowed to ensure that the water had time to “lyse” all of the cells, and then the contents were centrifuged for 15 minutes at 3000 rpm, and 13( C. When this process was complete, a liquid solution of hemoglobin resulted that was significantly less viscous than the original sample of blood. The cells that had settled at the bottom of the tube appeared brownish. The hemoglobin (aq) was poured into another test tube(s) and the settled cell bodies were discarded. Hemoglobin had now been successfully extracted from the raw blood sample.

For some of the experiments that we performed it is helpful to know the molarity of the sample of hemoglobin, but this is difficult to determine due to the inexact methods used when washing and lysing the cells. A convenient method is to assume that the hemoglobin that comes from the lysing process has a molarity of 1.875 x 10-3 (calculated in appendix E) we can then use the equation:

M1V1 = M2V2

Where the M1 and M2 represent the initial and final molarities respectively, and the Volumes are the respective volumes of the hemoglobin lysed and the hemoglobin that has been diluted. Through this method we were able to determine a relatively accurate molarity for the hemoglobin that we entered into the Spectrophotometer.

Dilution and Working Solution of Hemoglobin

Since the maximum absorbance reading of the Milton Roy Spectronic 20 Spectrophotometer is 2.0, an adequate dilution of the hemoglobin solution was prepared with a maximum value that fell within the range of 1.2 to 1.5. This range was chosen so that the absorbance reading for all wavelengths between 340-600nm. A suggested wavelength of 400nm (BE 210 Manual Spring 1996) was used to test the dilutions. For each of the four different stock hemoglobin samples prepared, the dilutions were 15x, 15x, 300x, and 300x, respectively. A total of 100 mL hemoglobin solution was prepared for the first two samples and 50 mL for the last two samples. The appropriate amount of hemoglobin sample was pipetted in to the corresponding volumetric flask which was then filled up to the mark with deionized water. An assortment of pipettes including the Pipet-Aid, 20-200(L, and 100-1000(L pipettes were used to make precise measurements of hemoglobin. This working solution of hemoglobin was now poured into two beakers.

One beaker was used as the working oxyhemoglobin solution. This solution was stirred to ensure a pure form of hemoglobin in the oxy form, and labeled as 100%. The appropriate pipettes were used to make dilutions of 80%, 60%, 40%, 20%, and 10%, 3 mL of each which were placed into 13 mm cuvettes in a test rack and covered with parafilm.

The second beaker was used as the working deoxyhemoglobin solution by adding sodium dithionite and stirring. Sodium dithionite is a white crystal powder with a slight odor. This chemical oxidizes in air, although does so more readily in solution. It is very soluble in water and can be used as a reducing agent.( Merck Index) Oxygen that is attached to the heme groups binds with this chemical, resulting in deoxyhemoglobin. Small quantities of the chemical were added at a time to ensure complete dissolution of the mixture. Additions were added until the solution changed from a pale pink to bluish/purplish color. A sample of this was placed in a cuvette and tightly sealed with parafilm to ensure no oxygen entry and sodium dithionite escape. Dilutions of the deoxyhemoglobin solution were made similar to those of hemoglobin, although these were made later to avoid any oxygen contamination.

Calibration of the Spectrophotometer

The Spectronic 20 Spectrophotometer was turned on and allowed to warm up for 15 minutes. After the warm-up period, with the wavelength set at 400nm and the sample compartment empty, the Spec was zeroed at transmittance mode. Then, with a cuvette filled with deionized water, the meter was placed on absorbance mode and zeroed. Careful precautions were taken to make sure a clean cuvette (i.e. without fingerprints or water droplets) was placed into the compartment to ensure an accurate absorbance reading.

Determination of the Oxyhemoglobin Spectrum

Approximately 3 mL of the 100% oxyhemoglobin working solution was placed into a cuvette. This measurement need not be exact, only enough was needed to fill the cuvette to allow for an accurate reading. A “blank” was also ready in order to calibrate the meter to zero absorbance before each reading. For the first sample of working solution, the absorbance was taken starting at a wavelength of 350nm, and proceeded in 25nm increments up until the maximum wavelength of 600nm. Wavelength and absorbance values were recorded using the Labview program that was specifically designed for use with the spectrophotometer. A graph of absorbance vs. wavelength was constructed. From this spectrum, the highest wavelength peaks were defined, and additional measurements were taken at smaller wavelength increments (3-5nm) to more accurately determine the peak. Starting from a wavelength about 25nm below the apparent peak and then gradually increasing the wavelength, a maximum absorbance was reached. Three apparent peaks were determined for the oxyhemoglobin, but the highest peak was the peak of concern. For the three other working solutions, testing involved finding this highest peak.

Determination of the Deoxyhemoglobin Spectrum

Identical procedures were carried out for the determination of the deoxyhemoglobin spectrum. Rather than working with both oxy- and deoxyhemoglobin at the same time, each peak was determined separately. This was to avoid any oxygen contamination of the deoxygenated hemoglobin.

Concentration Dependence

For each of the four working solutions of oxy- and deoxyhemoglobin, a concentration vs. Absorbance curve was constructed. The meter was set at the determined highest peak wavelength for each set of readings. The prepared diluted solutions were then measured for absorbance level. These values were then graphed.

Determination of Oxy/Deoxyhemoglobin Spectrum

For hemoglobin samples 3 and 4,(the second sample was used up), a 100% working solution was prepared both for oxy- and deoxyhemoglobin using the procedure above. Starting with 100% oxyhemoglobin, dilutions of 80%, 60%, 40%, 20%, and 0% were made with the other percentage consisting of deoxyhemoglobin rather than deionized water. A spectrum of each dilution was then created to find the peak wavelengths. As before, absorbance readings were taken starting at a wavelength of 350nm, and proceeded in 25nm increments up until the maximum wavelength of 600nm. Once the highest peak wavelength was determined, the two other trials concentrated only around this wavelength. A graph of absorbance vs. wavelength was plotted. A paired t-test was used for error analysis and test of significance between the two results.

Results

The objective of the first part of our project was to determine the absorption spectrums of oxy- and deoxyhemoglobin. Graphs 1 and 2 are the plots of the two spectrums. From the two graphs, we were able to determine smaller ranges in which their peak wavelengths and absorbances occurred. The error in all of the absorbance readings was 0.005.

Graph 1: Absorbance Spectrum for Oxyhemoglobin

[pic]

Graph 2: Absorbance Spectrum for Deoxyhemoglobin

[pic]

In Table 1, the ranges of the peak wavelengths for the two types of hemoglobin are displayed. Because the oxyhemoglobin solution was red, we hypothesized that it would predominantly absorb wavelengths in the violet region of the visible spectrum (approximately 400 nm). In accordance with our hypothesis, the greatest peak for the oxyhemoglobin was between 405 and 410 nm. Less prominent peaks for the oxyhemoglobin were at 545 and 555 nm respectively. The deoxyhemoglobin also showed a principal peak in this region, but it peaked at the wavelength of 410 nm exclusively. This peak can be seen in Graph 2 and Table 1. This change in wavelength could be attributed to the slightly purplish tint the solution took on when the oxygen was removed from the mixture. Since the solution was emitting some purple wavelengths, fewer wavelengths in this color range could be absorbed. As expected the less prominent wavelengths for the deoxyhemoglobin were also slightly higher than those for the oxyhemoglobin; they occurred at 550 and 575 nm, respectively.

Table 1: Peak Absorbances

| |Peak (1 |Absorbance |Peak (2 |Absorbance |Peak(3 |Absorbance |

|Oxy-hemoglobin | | | | | | |

| |405 - |1.9 +/- .005 |545 nm |0.222 |555 nm |0.192 |

| |410 nm | | |+/- .005 | |+/- .005 |

|Deoxy- | | | | | | |

|hemoglobin |410 nm |1.13 +/- .005 |555 nm |0.205 |575 nm |0.167 |

| | | | |+/- .005 | |+/- .005 |

Our next goal was to verify exact wavelengths of maximum absorbance for the oxy- and deoxyhemoglobin. In order to do this we chose an abridged spectrum of wavelengths to test. The oxyhemoglobin peaked in a range from 405 to 410 nm, thus we decided to test its absorbance over a range of wavelengths spanning from 350 to 450 nm. The deoxyhemoglobin peaked at 410 nm, which was slightly higher than the oxyhemoglobin, so we decided to test its absorbance from 375 to 450 nm. To ensure our experiment was void of flaws due to human errors in extracting the hemoglobin from the horse blood, we utilized three different hemoglobin samples and did three trials for each sample. Our results will be exemplified using our best trial, hemoglobin #2. While the other two trials were factored into our final results, the data and analysis of each are placed in Appendices A and C.

The absorbance spectrum of the oxyhemoglobin from sample 2, can be seen in Graph 3. In this graph, the outcome of the three trials are superimposed to show the great extent to which they coincide. Although the greatest absorbances range from 1.31 to 1.70, due to the differences in the concentrations of the hemoglobins, the optimal wavelength for absorption remains the same in all three, 410 nm. The error in absorbance readings for all of the trials, is 0.005 which is the inherent error in the spectrophotometer.

Graph 3: Oxyhemoglobin Peak (Hemoglobin #2)

[pic]

After comparing the results from this hemoglobin sample to those from the two other samples, we found that while the peak absorbances varied from a low of 1.43 to a high 1.92, the wavelength where this peak occurred remained the same for all nine trials, 410 nm. The data for the oxyhemoglobin trials, along with the actual wavelength of peak absorbance for horse-blood can be seen in Table 2.

Table 2: Peak Wavelengths for Oxyhemoglobin

| |Wavelength (nm) |(mean) Absorbance |

|Hemoglobin 1 |410 |1.92 +/- .005 |

|Hemoglobin 2 |410 |1.49 +/- .005 |

|Hemoglobin 3 |410 |1.43 +/- .005 |

|Actual Horse Hemoglobin |412 |- |

From Table 2, we calculated the percent error between the actual peak wavelength for horse oxyhemoglobin and our experimental value for horse oxyhemoglobin to be 0.485%.

Once the procedure was completed for oxyhemoglobin, we then repeated it for the deoxyhemoglobin. We wanted the experiment to have the exact same controls and people conducting it, in order to eliminate human error as a source of difference in their peak wavelengths. Once again, the second hemoglobin sample is used to demonstrate our results. Graph 4, indicates that while the maximum absorbance values differed from 1.5 to 1.56, the wavelength at which these values occurred was the same for all three trials, 421 nm.

Graph 4: Deoxyhemoglobin Peak (Hemoglobin #2)

[pic]

We then compared the mean values for wavelengths and absorbances to those for the two other hemoglobins. As can be seen in Table 3, just as the nine trials for oxyhemoglobin peaked at an identical wavelength, so did the nine trials for deoxyhemoglobin. The variance in absorbances can again be attributed to differences in dilutions of hemoglobin.

Table 3: Peak Wavelengths for Deoxyhemoglobin

| |Wavelength (nm) |(mean) Absorbance |

|Hemoglobin 1 |421 |1.78 +/- .005 |

|Hemoglobin 2 |421 |1.54 +/- .005 |

|Hemoglobin 3 |421 |1.42 +/- .005 |

|Actual Horse Hemoglobin |430 |- |

While the deviance between the value for the actual horse oxyhemoglobin and our experimental value was only 0.485 %, the deviance for the deoxyhemoglobin was slightly higher. We determined that our samples varied from the true values by 2.093 %. This value exemplifies a very insignificant amount of error, i.e. it is less than 5 %,

In the second part of the experiment, our objective was to find the molar extinction coefficients for the two hemoglobins at their peak-absorbance wavelengths. We again utilized the three hemoglobins and conducted three trials on each in order to find a mean coefficient. To find values for the coefficients, the concentrations of our solutions needed to be known. To determine the concentrations, we utilized literature values for the density and molecular weight of horse hemoglobin. Since the density is 120 grams / Liter, and the molecular weight is 64000 grams / mole, the molarity of the original hemoglobin was 1.875 * 10-3 moles / Liter. We then added 1 ml of hemoglobin to 50 ml of water, by utilizing the equation: M * V = M * V (See Background), we found the new concentration to be 3.75 * 10-5. In order for the absorbances to be able to be read by the spectrophotometer, in other words under 2.00, the concentrations had to be on the order of 10-6, thus we made dilutions of 15x and 300x to render concentrations in this range. After making these dilutions, our 100% solutions were 2.5 * 10-6 moles / Liter and 1 * 10-6 moles / Liter. The rest of the concentrations are calculated in Appendix E and F. Our procedure will be exemplified using the data from the second hemoglobin trial which has an initial concentration of 1 * 10-6. Graph 5 is the plot of Absorbance vs. Percent Oxyhemoglobin Concentration at its peak wavelength of 410 nm. (The other six trials can be seen in Appendices A and C).

Graph 5: Oxyhemoglobin Absorbance vs. Percent Concentration

[pic]

From Graph 5, it is possible to see that the first two trials of hemoglobin #2, rendered almost the exact same results because the graphs are nearly collinear, while the third trial must have utilized a more dilute sample of oxyhemoglobin, thus producing lower absorbance readings and a line of lower slope. The R2 Coefficient is 0.9996, thus exemplifying that the relationship between absorbance and percent concentration for oxyhemoglobin is almost perfectly linear. Hence as the concentration of the oxyhemoglobin increased, so did its absorbance levels. Once we established the linearity between the two properties, we needed to find the slopes of the lines for each of the trials. Utilizing the equation, A = ( * C * l (see Background)

the slopes of the three lines could be taken to be “e”, the molar extinction coefficient. By doing a linear regression on each of the three lines (an example is given for the first trial), and completing a statistical analysis on the individual points (See Appendices A and C) a mean slope was found to be 1.435 +/- 0.076. Since the concentrations were on the order of 10-6 moles / Liter to get absorbance readings between 0 and 2 (See Appendix E for the concentrations correlated with the different percentage values), the molar extinction coefficients had to be on the order of 105. Hence for the second sample of hemoglobin, the molar extinction coefficient for oxyhemoglobin at a wavelength of 410 nm was determined to be 14.35 *104 L / mol * cm. Table 4 shows the mean coefficients for the three trials along with the actual value for horse oxyhemoglobin at a wavelength of 410 nm.

Table 4: Molar Extinction Coefficients (Oxyhemoglobin)

(The Wavelength is 410 nm)

| |Molar Extinction Coefficient |

| |(L / mol * cm) |

|Hemoglobin 1 | 18.9 * 104 +/- 0.064 |

|Hemoglobin 2 | 14.5 * 104 +/- 0.076 |

|Hemoglobin 3 | 14.4 * 104 +/- 0.105 |

|Mean for the 3 | |

|Hemoglobins |15.9 * 104 +/- 0.082 |

|Actual Coefficient at | |

|412 nm |12.4 * 104 |

After finding a mean molar extinction coefficient for the three trials of the first hemoglobin sample, we did the same for the other two and then averaged the three values. We then contrasted this experimental value, 15.9 * 104, to the actual coefficient at 412 nm, which is12.4 * 104. In doing this we found that ours was divergent from the value at 412 nm by 28.47%.

In order to compare our molar extinction coefficient at 410 nm to the actual coefficient at that wavelength, we would have had to have utilized a constant, known concentration of hemoglobin for all nine trials. Because we centrifuged the samples from the blood ourselves, we were not sure of the exact concentration of hemoglobin that was present before we made the dilutions. Hence we could not utilize the equation:

A = ( * C * l

to calculate the exact coefficient at each of the absorbances. To compensate for this inadequacy, though, we employed a plot of molar extinction coefficients vs. wavelength for hemoglobin and oxyhemoglobin from the book Hematin and Bile Pigments by Lemberg and Legge (See Appendix F).

Once we completed our analysis of oxyhemoglobin, we then performed the same analysis on the deoxyhemoglobin at its peak wavelength of 421 nm. Graph 7 is the plot of Deoxyhemoglobin Absorbance vs. Percent Concentration for the second hemoglobin sample.

Graph 7: Deoxyhemoglobin Absorbance vs. Percent Concentration

[pic]

For the deoxyhemoglobin, a sample linear regression is once again provided. This data with a R2 coefficient of 0.9982, supports a linear relationship between absorbance and percent concentration of deoxyhemoglobin that is even more assured than the data for oxyhemoglobin. By comparing the linear regressions and statistical analyses of the three sets of data, a mean molar extinction coefficient for this sample of deoxyhemoglobin was found to be 15.4 * 10^4 L / mol * cm. The mean molar extinction coefficients for the other two samples, along with the actual coefficient at 421 nm can be found in Table 5.

Table 5: Molar Extinction Coefficients (Deoxyhemoglobin)

(The wavelength is 421 nm)

| |Molar Extinction Coefficient |

| |(L / mol * cm) |

|Hemoglobin 1 |16.1 * 104 +/- 0.193 |

|Hemoglobin 2 |15.4 * 104 +/- 0.125 |

|Hemoglobin 3 |13.8 * 104 +/- 0.135 |

|Mean for the 3 | |

|Hemoglobins |15.1 * 104 +/- 0.151 |

|Actual Coefficient at | |

|420 nm |11.5 * 104 |

For each of the samples of deoxyhemoglobin, we again found a mean coefficient, then averaged the three to find a coefficient for the entire deoxyhemoglobin experiment. We compared our coefficient of 15.1 * 104 L / mol *cm to the accepted value at 430 nm, 11.5 * 104, and determined that ours varied from the actual value by 31.30 %. We were able to employ the same reasoning for utilizing the extinction coefficient at the actual peak wavelength as a comparison because as was with the case of the oxyhemoglobin, 421 nm is within +/- 2.5 nm of the actual peak wavelength of 420 nm.

To find the uncertainties in our concentration values, it was necessary to add all of the uncertainties in each of the pipettes used to make the dilutions. Thus for all of our 15x dilutions, the error was the same. Table 6a is the error induced by each of the three types of pipettes and Table 6b is an explanation of the uncertainty values for each of our concentrations of oxy- and deoxyhemoglobin.

Table 6a: Error Due to Measuring Instruments

|Instrument |Error (in ml) |

|1 ml Pipette |0.001 |

|0.2 ml Pipette |0.0001 |

Table 6b: Uncertainty Intervals for 15x and 300x Dilutions

|Concentration |Solution |Uncertainty |Water |Uncertainty |Total Uncertainty |

|100 % |3.0 ml |0.003 |0.0 ml |0 |0.003 ml |

| 80 % |2.4 ml |0.002 |0.6 ml |0.001 |0.003 ml |

| 60 % |1.8 ml |0.001 |1.2 ml |0.001 |0.002 ml |

| 40 % |1.2 ml |0.001 |1.8 ml |0.001 |0.002 ml |

|20 % |0.6 ml |0.001 |2.4 ml |0.002 |0.003 ml |

|10 % |0.3 ml |0.001 |2.7 ml |0.002 |0.003 ml |

Even though, the oxyhemoglobin and deoxyhemoglobin rendered different molar extinction coefficients, the result of the two experiments was the same. They both verified that there is a linear relationship between hemoglobin absorbance and concentration, and that the relationship is independent of oxygenation.

For the last part of the experiment, we utilized the results of the first two parts as a baseline. We previously determined that the oxyhemoglobin exhibited a peak absorbance at 410 nm and deoxyhemoglobin exhibited a peak absorbance at 421 nm. We also concluded that for the two types of hemoglobin, absorbance is directly proportional to the concentration, thus for changing concentrations, the peak absorbances will change in a linear fashion. By combining these two facts, with the idea that as the deoxyhemoglobin is added to oxyhemoglobin in equal intervals, the color of the oxyhemoglobin will change proportionately, we hypothesized that the peak wavelengths should move from 410 nm at 100% oxygen to 421 nm at 0% oxygen (100% deoxyhemoglobin) in a linear fashion. The latter linear transition can be attributed to the changing of color, and in turn absorbance, of the solutions as more deoxyhemoglobin is added. We attempted to substantiate our hypothesis by conducting two trials with different samples of hemoglobin. Graphs 8a and b are plots of our results.

Graph 8a: Peak Wavelength vs. Percent Oxyhemoglobin

[pic]

Table 7: Peak Wavelengths for Oxy - Deoxyhemoglobin Mixtures

|%Concentration |Peak Wavelengths (nm) |Average Peak Wavelength (nm) |

|100 |410 |410 |

|80 |410 - 411 |410.5 |

|60 |412 - 414 |413 |

|40 |413 - 415 |414 |

|20 |418 - 419 |418.5 |

|0 |421 |421 |

Graph 8b: Average Peak Wavelength vs. Percent Oxyhemoglobin

[pic]

From Graph 8a, many things about the relationship between percent oxygen concentration and peak wavelength can be inferred. First of all, only the 0% and 100% oxygen concentrations render a single peak wavelength. The other concentrations all have either two or three consecutive wavelengths where a peak absorbance occurs (See Table 6). As expected, though, the 0% oxyhemoglobin (100% deoxyhemoglobin) climaxes at 421 nm and the 100% oxyhemoglobin (0% deoxyhemoglobin) climaxes at 410 nm. The peak wavelengths for each of the concentrations in between the maximum and minimum concentrations are also in between the latter maximum and minimum wavelengths. In fact, as the percent deoxyhemoglobin decreased and the percent oxyhemoglobin increased, the peak wavelength moved nearly linearly from the peak for deoxy- to the peak for oxy-, from 421 to 410 nm respectively. Graph 8b, which is the plot of the Average Peak Wavelength vs. Percent Oxyhemoglobin, has an R2 coefficient of, 0.9426, which strongly supports the linear, inverse relationship between the optimal wavelength of absorbance and the percent oxyhemoglobin. It is obvious from this graph, that as the amount of oxygen in the hemoglobin decreases, the peak wavelength of absorbance increases.

Discussion

After having finished the calculations and error propagation, we looked at our results to find qualitative reasons for their values. We found that there was indeed a peak wavelength at which the oxyhemoglobin and deoxyhemoglobin have maximum absorbance. Knowing the peak wavelength and molar extinction coefficients, we were able to calculate the concentration. The determination of the peak wavelengths were found to not be significantly different from the literature values. The oxyhemoglobin and deoxyhemoglobin had an error of 0.485% and 2.093%. This is well within our 95% confidence interval. In addition to this final calculated percent error we are also confident in our experimental values because we completed three trials on three separate hemoglobin extractions.

Our values for the molar extinction coefficients, however, were off by approximately 30%. For the oxyhemoglobin and deoxyhemoglobin we obtained percent errors of 28.47% and 31.30%. This error is not exact because we did not have exact literature values of the molar extinction coefficients at our peak wavelengths. Rather, the molar extinction coefficient values were compared to literature values at 412nm for the oxyhemoglobin and 420nm for the deoxyhemoglobin. Our calculated molar extinction coefficients were determined from our experimental peak wavelength which was 410nm for the oxyhemoglobin and 421nm for the deoxyhemoglobin. Another factor that could have caused the great difference was our assumption that the extracted hemoglobin was 100% pure hemoglobin. We had no way of determining what exact percent concentration at the time of extraction.

In determining the peak wavelengths of oxy- and deoxyhemoglobin, there are several sources of error that we tried to avoid. There are three main categories of errors. These are illegitimate, random, and systematic. Illegitimate errors are due to sloppy experimental techniques and normally accounts for the greatest difference between experimental values. We virtually eliminated this error by continuously checking each step in the procedure and making sure that it adhered to our outlined protocol. We also developed a standard habit of washing all of our glassware with soap, rinsing with deionized water, and drying them with kimwipes.

Next, are random errors. These types of errors are the ones that can be statistically analyzed since they most often arise out of measurement errors. The errors are inherent in the instruments that were used in the lab. To reduce as much instrumental errors as possible, we measured the deionized water using a 10 +/-0.1ml pipette. For volumes less than 1ml, micro-pipettes were used instead, which lowers the sources of error from +/-0.1ml to +/-0.001ml. Finally, the other source of error is the spectrophotometer which gave outputs that fluctuated intermittently. Although three readings were made for each sample and an average was computed, the fluctuations still contributed errors of up to +/- 0.001 Absorbance reading. All of these errors then affect the final computed experimental values for the peak wavelengths and molar extinction coefficients.

Finally, there are several sources of systematic errors. These errors are often consistent throughout the experiment, but in the end they can change the final experimental value so that it is not very accurate. They usually stem from limitations in the instruments, environmental variations, and random fluctuations in the experimental process. First, is the clarity and cleanliness of the cuvettes. Some of them have scratches and/or dried impurities from previous solutions that can diffract or block the monochromatic beam of light in the spectrophotometer. To reduce this error as much as possible we washed the cuvettes with soap and deionized water, and dried them with kimwipes. We then checked each cuvette and chose those that are relatively free of defects and impurities. Before inserting the cuvettes into the spectrophotometer we wiped the sides once more with kimwipes and made sure that they were aligned correctly. Once we cleaned the cuvettes, we attempted to find the error that was inherent in the spectrophotometer itself. In order to do this, we filled 10 curettes with deionized water, and set the absorbance reading for the first cuvette to zero. We then determined the absorbances of the subsequent nine curettes, relative to the first cuvette and found the average of their readings. The mean value of +/- 0.001 was then used as the uncertainty interval for all of our absorbance readings. The second source of error that we tried to eliminate was the accidental zeroing of the spectrophotometer. We made sure that each time we changed wavelengths on the spectrophotometer, we zeroed the absorbance readings with deionized water. Next are the errors that can arise from air bubbles forming in the solution and deionized water. After a long period of time small bubbles tend to form in the cuvettes. By tapping each of the cuvettes we were able to accomplish two things, remove bubbles and mix the solution. This process ensured that the solution is uniform and free of bubbles that can diffract light. Finally, we tried to ensure consistent procedural protocol by having the same person perform the same duties for each trial.

After calculating the peak wavelengths of both oxy- and deoxyhemoglobin we conducted a third experiment. This experiment involved mixing two 100% solutions of oxyhemoglobin and deoxyhemoglobin in different percent concentrations. Measurements were taken with concentrations of 100%, 80%, 60%, 40%, 20%, and 0% of the oxyhemoglobin that was mixed with a corresponding amount of deoxyhemoglobin. Our results show that as oxyhemoglobin concentrations decreases the wavelength increases. This supports our hypothesis of being able to determine the concentration of oxy/deoxy hemoglobin in a sample. However, we must also take into account that we used sodium dithionite to make our deoxyhemoglobin in imprecise amounts. Since, we did not know the exact amount of reducing agent that was used, there could have been excess in the deoxyhemoglobin solution that was not bound to oxygen. Once the two solutions were mixed, the sodium dithionite could have bound with the oxygen in the oxyhemoglobin, thus converting it to deoxyhemoglobin. This could explain why our range fluctuated between several values instead of peaking at a single point. In order to avoid this mistake in future experiments, a more precise method of deoxygenation should be used such as bubbling nitrogen through the oxyhemoglobin solution. Also the mixing of the two hemoglobin solutions should be conducted in an anaerobic environment, so oxygen from the air will not permeate the solutions and skew the results.

Finding the peak wavelength of oxyhemoglobin and deoxyhemoglobin is important because it provides a method in analyzing the concentration levels of hemoglobin in a given blood sample. Often times there are other dangerous chemicals that can take the place of oxyhemoglobin and thus block the passage of oxygen molecules to the cells in the body. One such chemical is carbon monoxide. Carbon monoxide, when bound to hemoglobin, has the similar characteristic color of cherry red and peaks at approximately the same wavelength. In the future, better spectroscopy techniques can be developed so that we can detect and analyze the differences between oxyhemoglobin and carbon monoxide hemoglobin. Hospitals are already using similar techniques in helping doctors diagnose patients who seem to have low levels of oxygen in their blood. In conclusion, spectrophotometry is a valid method in determining the concentrations of oxy- and deoxyhemoglobin levels in a sample of blood.

Works Cited

BE 210 Lab Manual. Dept. of Bioengineering, University of Pennsylvania: Philadelphia, Spring 1996.

Biophysical Journal. 70(4): 1949-65. April, 1996.

Brickman, Dr. Kris, President, Emergency Services. Medical College of Ohio, Toledo, Ohio. Phone interview, March 18, 1997.

Bunn, H. Franklin, MD., Bernard G. Forget, MD., and Helen M. Ranney, MD. Human Hemoglobins. W. B. Saunders Company: Philadelphia, 1977.

Callister, William D., Jr. Material Science and Engineering: An Introduction. John Wiley & Sons, Inc. 4th ed. New York: 1997.

“Crystal Structure of HB.T in deoxy and carbonmonoxy forms” Journal of Molecular Biology. 259(4):749-60. June 21, 1996.

Journal of Occupational and Environmental Medicine. 38(4):367-71. April 1996.

Lemberg, R and Legge, J.W. Hematin compounds and bile pigments. New York: Interscience Publishers 1949.

Meggers, William Frederick, Charles H. Corliss, and Bourdon F. Scribner. Tables of spectral-line intensities. National Bureau of Standards. 2d ed. 1975.

Stryer, Lubert. Biochemistry. W.H. Freeman and Company, Fourth Edition. 1995.

Windholz, Martha, ed. The Merck Index. 9th ed. New Jersey: Merck and Co. 1976.

Appendix

|Appendix A: | | | | | | | |

|Hemoglobin 1 | | | | | | | |

| | | | |mean | | |1.916667 |

| | | | |absorbance | | | |

|Oxyhemoglobin | | | | |

|Peaks | | | | |

| | | | | |

[pic]

|Oxyhemoglobin | | |

|Dilutions | | |

[pic]

|Deoxyhemoglobi| | |

|n 1 | | |

[pic]

|Deoxyhemoglobin | | | |

|Dilutions | | | |

[pic]

|Appendix B: | | | |

|Hemoglobin 2 | | | |

| | | | |

|Oxyhemoglobin 2 | | | |

[pic]

|Oxy Dilutions | | | |

| | | | |

The wavelength is 410 nm.

[pic]

|Deoxyhemoglobin 2| | | |

[pic]

|Deoxy dilutions | | | |

| | | | |

|The wavelength is | | | |

|421 nm | | | |

[pic]

|Appendix C: | | | |

| | |Hemoglobin 3 | |

| | | | |

|Trial 1 | | | |

[pic]

|Hemoglobin 3 Oxy | | | |

|Dilutions | | | |

| | | | |

[pic]

| | | | |

| | | | |

| | | | |

| | | | |

|Hemoglobin 3 | | | |

|Deoxyhemoglobin | | | |

| | | | |

| | | | |

[pic]

|Hemoglobin 3 Deoxy Dilutions | | | | | | | |

[pic]

|Appendix D | | |

|100% | | |

|Oxyhemoglobin | | |

| | | |

[pic]

|80% Oxyhemoglobin| | | | | |

|- 20 % | | | | | |

|Deoxyhemoglobin | | | | | |

[pic]

| | | | | | | |

| | | | | | | |

| | | | | | | |

| | | | | | | |

| | | | | | | |

|60% | | | | | | |

|Oxyhemoglobin | | | | | | |

|- 40% | | | | | | |

|Deoxyhemoglobi| | | | | | |

|n | | | | | | |

| | | | | | | |

[pic]

|40% Oxyhemoglobin| | | | | | |

|- 60% | | | | | | |

|Deoxyhemoglobin | | | | | | |

| | | | | | | |

[pic]

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

| | | | | | |

|20 % | | | | | |

|Oxyhemoglobin - | | | | | |

|80 % | | | | | |

|Deoxyhemoglobin | | | | | |

| | | | | | |

[pic]

|0 % Oxyhemoglobin| | | | | | |

|- 100 % | | | | | | |

|Deoxyhemoglobin | | | | | | |

| | | | | | | |

[pic]

|Appendix E | |Concentration | | |15 x | | |

| | |Calculations | | | | | |

| |Density of | | | | |120 g/L | |

| |Horse | | | | | | |

| |Hemoglobin | | | | | | |

| |Molecular | | | | |64000 g/mol | |

| |Weight of | | | | | | |

| |Horse | | | | | | |

| |Hemoglobin | | | | | | |

| |Molarity of | | | | |.001875 | |

| |Horse | | | | |mol/L | |

| |Hemoglobin is:| | | | | | |

| |If we start | | | | | | |

| |originally | | | | | | |

| |with 1 ml of | | | | | | |

| |horse | | | | | | |

| |hemoglobin | | | | | | |

| |(after | | | | | | |

| |centrifuging) | | | | | | |

| | |and dilute it with 50 | | | | | |

| | |ml of water. | | | | | |

| |By using the | | | | | | |

| |equation: | | | | | | |

| |M1*V1 = M2*V2 | | | | | | |

| | | | | | | | |

| |The original | | | | |3.75E-05 |mol/L |

| |concentration | | | | | | |

| |of hemoglobin | | | | | | |

| |that we use is| | | | | | |

| |: | | | | | | |

| | | | | | | | |

| |We then made | | | | | | |

| |15x dilutions.| | | | | | |

| |So our 100% | | | | | | |

| |dilution was | | | | | | |

| |3.75*10^-5 / | | | | | | |

| |15. | | | | | | |

| | | | |2.5E-06 | | | |

| | | | | | | | |

| |To find each | | | | | | |

| |additional | | | | | | |

| |Concentration,| | | | | | |

| |we multiplied | | | | | | |

| |the 100% | | | | | | |

| |concentration | | | | | | |

| |by that | | | | | | |

| | | |fraction. | | | | |

| | |ie. 2.5*10^-6 * .8 | | | | | |

| | |for 80% | | | | | |

| | | |% Concentration |Concentration | | | |

| | | |100 |0.0000025 | | | |

| | | |80 |0.000002 | | | |

| | | |60 |0.0000015 | | | |

| | | |40 |0.000001 | | | |

| | | |20 |0.0000005 | | | |

| | | |10 |0.00000025 | | | |

For the 300x Dilutions

| | | | | | | | | |

| |If we start | | | | | | |

| |originally | | | | | | |

| |with 8 ml of | | | | | | |

| |horse | | | | | | |

| |hemoglobin | | | | | | |

| |(after | | | | | | |

| |centrifuging) | | | | | | |

| | |and dilute it with 50 | | | | | |

| | |ml of water. | | | | | |

| |By using the | | | | | | |

| |equation: | | | | | | |

| |M1*V1 = M2*V2 | | | | | | |

| | | | | | | | |

| |The original | | | | |0.0003 |mol/L |

| |concentration | | | | | | |

| |of hemoglobin | | | | | | |

| |that we use is| | | | | | |

| |: | | | | | | |

| | | | | | | | |

| |We then made | | | | | | |

| |300x | | | | | | |

| |dilutions. So| | | | | | |

| |our 100% | | | | | | |

| |dilution was | | | | | | |

| |3.75*10^-5 / | | | | | | |

| |15. | | | | | | |

| | | | |0.000001 | | | |

| | | | |0.001875 | | | |

| | | | | | | | |

| |To find each | | | | | | |

| |additional | | | | | | |

| |Concentration,| | | | | | |

| |we multiplied | | | | | | |

| |the 100% | | | | | | |

| |concentration | | | | | | |

| |by that | | | | | | |

| | | |fraction. | | | | |

| | |ie. 1.0*10^-6 * .8 | | | | | |

| | |for 80% | | | | | |

| | | | | | | | |

| | |% Concentration |Concentration | | | | |

| | |100 |0.000001 | | | | |

| | |80 |0.0000008 | | | | |

| | |60 |0.0000006 | | | | |

| | |40 |0.0000004 | | | | |

| | |20 |0.0000002 | | | | |

| | |10 |0.0000001 | | | | |

| | | | | | | | |

Appendix F: Comparisons to Actual Values

Although the units on Graph F are different than the ones we used in our graphs, the extinction coefficients in Graph F are in milli-Molars, while ours are in Liters / mole * centimeter, the essence of the graph is the same. The molar extinction coefficient remains at a peak value for at least 5 nanometers, thus we can infer that the molar extinction coefficient will be the same from 407 to 417 nanometers.

Graph F: Molar Extinction Coefficients vs. Wavelength

(Horse Hemoglobin)

[pic]

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