Experiment 10 10-1

BLOOD AND HEMOGLOBIN Overview In this lab you will learn about some of the roles of metal ions in biology, and you will specifically learn about the role of iron in hemoglobin, a protein that contains iron (Fe) and carries oxygen (O2) from the lungs to the tissues of our bodies. You will be introduced to the structure and function of hemoglobin through a method called spectrophotometry. This method will be used to measure the absorbance of blood samples from several different sources. Using this method, you will be able to determine the amount of hemoglobin in a blood sample. You will also chemically produce different forms of hemoglobin and understand how they are interconverted. In addition, you will learn about some medical conditions that are caused by hemoglobin abnormalities, and do an experiment designed to illustrate the treatment for one of these diseases.

A single subunit of hemoglobin

A. Introduction

Bioinorganic Chemistry Bioinorganic chemistry is the study of metal ions in biology. Hemoglobin, which you will be studying in lab contains iron (Fe), a transition metal. Many other enzymes, proteins, and vitamins require metal ions to function:

Table 1. Some roles of metal ions in biology. Metal Ion Protein or vitamin Function

Fe hemoglobin Transport of O2 in the body Fe myoglobin Delivers O2 to some muscles Co Vitamin BB12 important for nerve cell function and blood

formation Cr glucose tolerance factor facilitates glucose uptake Cu superoxide dismutase antioxidant Zn DNA polymerase synthesizes DNA Se glutathione peroxidase antioxidant

Experiment 10 10-2 Fe is an extremely important transition metal in biology. It is required by almost every living thing and it helps to perform many different biological functions, such as:

1. transport of O2 to tissues 2. release of O2 to muscle cells (myoglobin) 3. acts as internal magnetic compass (in bacteria, birds, even humans) 4. cofactor to oxidation/reduction enzymes

Because Fe exists in two different oxidation states, the two can be interconverted by the addition or removal of an electron:

Fe2+ Fe3+-1 e-

+1 e-

oxidation

reduction

Oxidation = loss of electrons Reduction = gain of electrons

Iron deficiency can cause symptoms of diarrhea, headaches, apathy, poor cold tolerance, and weakness. Lack of Fe also causes an inability to use fat as fuel, and an inability to perform aerobic activities well. Special proteins in the intestine help us absorb Fe from the food that we eat. However, under normal conditions only 10-15% of the Fe in food is absorbed. For instance, although spinach is very rich in Fe, it has very low bioavailability (hard for our bodies to use) because of the presence of oxalate ions (C2O42-) which form insoluble Fe salts. Fe that is absorbed is transported to the bone marrow, where it is used to make new blood cells. We can enhance the amount of Fe that we absorb from our food by avoiding foods that contain tannic acid (found in tea, coffee, nuts, and fruits), and EDTA (a food additive). These compounds bind strongly to Fe and do not allow its absorption. Vitamin C, on the other hand captures Fe and forms a more soluble form which is easily absorbed into the body. So, remember to drink orange juice with your Fe fortified breakfast cereal! Functions of Blood The average human body contains 6 L of blood. Blood is composed of three main components, the red blood cells (erythrocytes), white blood cells, and the plasma. Blood performs the 8 functions listed below. The first two functions are performed by the red blood cells. The human body contains about 1012 erythrocytes (red blood cells) which, in turn, contain 950 g of hemoglobin. Functions 3-7 are performed by the plasma, and function 8 is performed by the white blood cells. 1) Carries O2 from the lungs to tissues 2) Carries CO2 from tissues to the lungs 3) Carries nutrients from the digestive system to tissues 4) Carries waste products from tissues to excretory organs 5) With its buffer system, it helps to maintain the pH of the body 6) Maintains a constant body temperature 7) Carries hormones from the endocrine glands to wherever they are needed 8) Fights infection

Hemoglobin Hemoglobin, a protein found in the red blood cells, contains Fe (iron) and serves as the principal transporter of molecular O2 in the blood of humans as well as many other animals. The hemoglobin picks up O2 in the lungs and transfers it to the tissues where it is used by cells. There are two major parts of hemoglobin:

Experiment 10 10-3

1) heme: a porphyrin with an Fe in the center. Porphyrin rings are found throughout biological systems and serve many different roles including photosynthesis in green plants, delivering O2 in muscles (myoglobin) and transporting O2 in blood (hemoglobin). A porphyrin is made of four pyrrole rings linked so that the N atoms point toward the center of the ring.

NNH

N HN

H3C

CH CH2 CH3

CH CH2

CH3

CH2CH2COOH

CH2

HOOCCH2

H3C

Protoporphyrin IX

NN

N N

H3C

CH CH2 CH3

CH CH2

CH3

CH2

CH2COOHCH2

HOOCCH2

H3C

Fe2+Fe2+

Ferroheme

N

H

A pyrrole ring

The four N atoms have lone pairs which can bind to metal ions such as Fe2+, Mg2+, etc. The porphyrin ring which is found in hemoglobin has a special group of substituents on the outside of the porphyrin ring (methyl, vinyl, and propanoic acid groups). This special type of porphyrin is known as Protoporphyrin IX. When Fe binds to Protoporphyrin IX, the unit is called a heme group. The Fe in hemoglobin can be in the ferrous (Fe2+) or the ferric (Fe3+) state. The Fe2+ version of heme is called ferroheme; ferroheme is the active, O2 binding form of the heme group.

The heme group is what gives the red color to hemoglobin. By changing the structure of the heme group, one can change the color of blood (you will be doing this in lab!).

2) The 2nd part of the hemoglobin is the globin. Globin is the protein (polypeptide) surrounding the

heme . The structure of the protein itself is discussed in more detail below.

Nomenclature: globin + heme (Fe2+) = hemoglobin globin + heme (Fe3+) = methemoglobin We use the prefix met to designate hemoglobin with Fe in the +3 oxidation state. While hemoglobin is the active form which can bind O2 and deliver it to the tissues, methemoglobin cannot combine reversibly with O2, and is not a useful form of the protein.

Structure of Hemoglobin The overall structure of hemoglobin consists of four polypepetide chains (globins) which are associated with one another. There are several different globin molecules with nearly identical spatial structure but who differ in their amino acid sequence. These globin chains are designated by a Greek letter – α, β, δ, ε, etc. Each globin molecule is combined with a heme group. Each globin and heme unit together is called a subunit. Each hemoglobin molecule contains four globin units with four heme units. Therefore, how many subunits are there in one hemoglobin?

Experiment 10 10-4 Adult human hemoglobin (HbA) has 2 equivalent halves, each containing an α subunit and a similar β subunit. Therefore, the overall structure of HbA is α2β2 since each hemoglobin molecule has 2 α and 2 β subunits. Sickle cell anemia, a dangerous condition in which red blood cells are very fragile and misshaped, is caused by a form of hemoglobin which has the same heme group, but a mutation in the amino acid sequence of one of the globin part of the protein. The animal hemoglobin that you will be investigating in this lab also differs slightly from the structure of human hemoglobin, but it is similar enough for the tests that you will be performing on it. Function of Hemoglobin Hemoglobin (Hb) transports O2 from the lungs to the tissues, where it releases the O2 and then returns to the lungs to pick up more O2. When hemoglobin is carrying O2 it is called oxyhemoglobin and when it is not carrying O2 it is known as deoxyhemoglobin. Deoxy- and oxyhemoglobin have different colors, thus explaining why blood in our arteries (oxyhemoglobin) is brighter red than the blood in our veins. Oxygen binds to the Fe of the heme group because it has lone pairs which can coordinate to the Fe. Hb + O2 HbO2 Hb + O2

LUNGS BLOOD TISSUES deoxyhemoglobin (Fe2+) oxyhemoglobin (Fe2+) deoxyhemoglobin (Fe2+)

Fe2+

N N

N NOO

Oxygen binding to the heme group of Hemoglobin

Because each hemoglobin protein possesses four subunits, each with its own heme group, it can combine with 4 02 molecules in the following four steps: Hb4 + O2 Hb4O2 (1 oxyHb subunit, 3 deoxyHb subunits) Hb4O2 + O2 Hb4O4 (2 oxyHb subunits, 2 deoxyHb subunits) Hb4O4 + O2 Hb4O6 (3 oxyHb subunits, 1 deoxyHb subunits) Hb4O6 + O2 Hb4O8 (fully oxygenated) The reaction of hemoglobin with O2 occurs very rapidly (in only 1/10 th of a second). Once one O2 molecule binds, the shape of the hemoglobin changes, making it easier for the next O2 molecule to bind, which makes it easier for the remaining 2 O2 molecules to bind to the hemoglobin. This happens in the lungs, and the erythrocytes (red blood cells) become saturated with O2 there. The hemoglobin transports the O2 to the capillaries where it is released for use in the tissues. In the capillaries, a molecule known as 2,3-BPG (2,3-bisphosphoglycerate) inserts into the deoxyhemoglobin to prevent O2 from binding again (so that the O2 released to the tissues stays in the tissues where it is needed). 2,3-BPG interacts with the deoxyhemoglobin, changing its shape so that it can't pick up O2.

Experiment 10 10-5

Effect of pH on the affinity for O2

The pH of the surroundings dramatically affects the affinity of hemoglobin for O2. In the lungs, pH is more alkaline, and hemoglobin picks up O2. Hemoglobin then transports O2 to the capillaries where the pH is more acidic (because of dissolved CO2 and lactic acid).

CO2 + H2O H2CO3 H+ + HCO3-

CO2 dissolved in the blood causes an increase in the acidity The additional H+ binds to the hemoglobin, changes its shape, and causes the hemoglobin to release its O2. The hemoglobin then picks up CO2 and transports it back to the lungs for expiration. However, the CO2 binds to the globin rather than at the heme group. Only a small change in pH (7.6 in lungs vs 7.2 in the tissues) results in this behavior.

HbO2 + nH+ Hb(H+)n + O2In the tissues, H+ causes the release of O2 from oxyhemoglobin

Carbon Monoxide Molecular oxygen is a small molecule with lone pair electrons. Because of this oxygen can form a coordinate covalent bond to the Fe of the ferroheme. This means that it donates both of the electrons that form the bond. Unfortunately, there are other small molecules that have lone pairs that can also bind to the Fe of hemoglobin, including carbon monoxide (CO). Carbon monoxide actually binds more strongly than O2 and only a small amount of it can severely decrease the oxygen carrying capability of hemoglobin (and eventually cause death). Treatment for such a problem includes giving large amounts of O2 to try to displace the poison.

O2 + HbCO + HbHbO2 + CO

HbO2HbCOHbCO + O2

CO displaces O2!!!!

Experiment 10 10-6 The Blue Fugates and Methemoglobin An unusual clinical problem involving hemoglobin is the case of the “Blue Fugates”. In an isolated valley in Kentucky, for almost 150 years, there have been reports of people descended from a French immigrant, Martin Fugate, and his American born wife, Elizabeth Smith, exhibiting skin colors in varying shades of blue. This puzzled much of the medical community. It was discovered by a doctor named Madison Cawein, that the “Blue Fugates” had a relatively high concentration of brown methemoglobin (Fe3+), the oxidized form of hemoglobin, which when viewed through the skin has a blue color. In normal people, hemoglobin (with Fe in the +2 oxidation state) is readily oxidized to the ferric (Fe3+) form, or methemoglobin, which is inactive toward oxygen transfer. Because this form is useless to the body, an enzyme, called diaphorase is used to reduce the methemoglobin back to normal ferrous (Fe2+) hemoglobin that can carry O2. In the Blue Fugates, however, a recessive gene caused unusually low levels of the enzyme diaphorase, increasing the level of methemoglobin in the blood, and resulting in a blue color when viewed through the skin. With an understanding of the cause of the problem, a remedy was found-the Blue Fugates were given pills of methylene blue, an organic dye. The methylene blue took the place of the enzyme diaphorase, turning the methemoglobin (Fe3+) back to hemoglobin (Fe2+) and giving them a normal pink rather than blue color. The deep blue dye, methylene blue (oxidized form) is first reduced in the body by pyridine nucleotides to give its colorless, reduced form. Then the methemoglobin oxidizes the reduced form back to the blue oxidized form. When the Fugates urinated after this treatment, they got quite a surprise! The blue form of the methylene blue was excreted in their urine, making it look like the blue color was just running out of their bodies! The Problem of Nitrites Another health problem that concerns the production of excess methemoglobin (Fe3+) is the problem of the effect of nitrite (NO2-) on hemoglobin. Nitrites can oxidize hemoglobin to methemoglobin, which cannot carry O2. Nitrites can be formed in our bodies from the reduction of nitrates (NO3-) which are common forms of pollution from fertilizer runoff. Methemoglobinemia, a disease resulting from this excess of methemoglobin is especially harmful to infants, because fetal hemoglobin is more easily oxidized. Nitrites are also added to many meat products to maintain the red color and to act as a preservative. You may be wondering why this is done since it was mentioned previously that nitrite causes oxidation to methemoglobin which is brown. However, if the nitrite is added to a solution containing hemoglobin and a reducing agent such as ascorbic acid, a red nitrosyl (NO) hemoglobin is formed (HbNO). In this form of hemoglobin, the NO group is bound to the Fe instead of O2. Thus, when both ascorbic acid (commonly added as a preservative) and NO2- are added to meat, a red color is produced. Nitrosylhemoglobin (HbNO) is more resistant to oxidation to Fe3+ over time than regular hemoglobin, and thus the meat stays red longer when it is on display.

Interaction of Pb2+ with Hemoglobin Pb2+ denatures hemoglobin (changes the tertiary structure of the protein) and prevents the Fe2+ from entering the porphyrin. Without Fe in the protein, it can't carry O2. Pb poisoning is a serious problem that leads to learning disabilities, low IQ, behavioral problems, and slow growth in children. It is also made more severe under conditions of Fe deficiency. Lack of Fe weakens the body's defenses against lead absorption. It is estimated that 1 out of 6 toddlers in the U.S. are exposed to high levels of Pb, mainly from lead water pipes and eating substances contaminated with lead (i.e. Pb paint or dust from environmental pollution).

Experiment 10 10-7 Spectrophotometry This technique utilizes a compound’s ability to interact with visible light to either absorb light or transmit light through a solution of the compound. Remember that white light is made up of all the colors of the spectrum. A solution which appears to be blue when viewed is actually removing or absorbing the red and green wavelengths of light and only allowing the blue light to be transmitted through the solution. The amount of light absorbed is a function of the wavelength of incident light; each compound will absorb light at different wavelengths and with different intensity. These characteristics allow us to compare compounds for identification purposes. The instrument that allows us to make these measurements is called a spectrophotometer. The spectrophotometer is made up of several parts:

1) A light source 2) A monochromator which separates light into separate wavelengths 3) A sample compartment which holds a cuvette (typically 1 cm wide) with the sample 4) A detector which converts light input into an output voltage 5) A recorder or computer for collecting the data for analysis

In this experiment, we will use the spectrophotometer to measure the absorbance of light at a fixed wavelength as it passes through a solution at a 1 cm pathlength. The ability to determine the concentration of a compound in solution is governed by the Beer-Lambert Law (Beer’s Law):

A= εcl Where A = absorbance (a unitless value – this is what the spectrophotometer will report), l = pathlength (typically 1 cm for modern instruments), ε = molar absorptivity coefficient (this is a constant for a given compound at a given wavelength – units of M-1cm-1), and c = concentration in molar units.

Therefore, absorbance is directly proportional to the concentration of the absorbing species in solution. By comparing the absorbance value obtained to known values it is possible to determine the concentration of the unknown sample. Measurement of Hemoglobin In order to determine the amount of hemoglobin in the blood, the hemoglobin must be released from the blood cells so that it is free in solution. The pigment is then measured using a spectrophotometer and the absorbance is compared to a series of standards that are used to draw a standard calibration curve. The most common method of this sort is to measure hemoglobin as cyanomethemoglobin. This method measures all types of hemoglobin in the body, including the oxidized form of hemoglobin (methemoglobin) which is inactive to O2, as well as oxy and deoxyhemoglobin. All of the hemoglobin is treated with potassium ferricyanide (K3Fe(CN)6 which contains Fe3+) and KCN. This ferricyanide/KCN solution has been used for many years for quantitative determination of hemoglobin and is called Drapkin's solution. The ferricyanide is an oxidizing agent: it contains Fe3+ which can oxidize the Fe2+ in oxyhemoglobin to Fe3+ and make the oxidized, met form of hemoglobin. (What happens to the Fe3+ in the ferricyanide?) The added free CN- (cyanide!) binds strongly to the oxidized form, resulting in cyanomethemoglobin. Cyanide forms a very strong, stable complex with methemoglobin, and this complex has an intense red color which absorbs at 540 nm in the visible spectrum. By measuring the absorbance of samples of hemoglobin and comparing them to known standards you will be able to determine the amount of hemoglobin in an unknown sample.

HbO2 (Fe2+) + Fe3+ (from ferricyanide) MetHb (Fe3+) + O2 + Fe2+

MetHb + CN- MetHbCN

Experiment 10 10-8 You will determine the amount of hemoglobin in blood samples of several different types of animals. Table 2 shows the normal hemoglobin levels in humans and a variety of animals. Notice that hemoglobin levels are typically given in g Hb/dL of blood. The levels of hemoglobin can vary among individual members of one species and among different species of animals. Reasons for this can include the diet, sex, and activity level of the animals.

Table 2. Hemoglobin levels for various species of animals Animal Hb (g/dL) Animal Hb (g/dL)

Dog 12-18 Horse (heavy breeds) (lightbreeds)

8-16 11-18 Cat 8-15

Ox 8-15 Rabbit 9-15 Sheep 9-16 Chicken 8-13 Goat 8-18 Turkey 11 Pig 10-18 Human 15

B. Procedure SAFTEY NOTES: 1) Since we are working with blood products, universal precautions must be observed. That

means that goggles and gloves should be worn through the course of this experiment. 2) We will be using cyanide solutions which are stable in the form that will be used in the

laboratory. However, it is very important to keep the cyanide solution away from acids – it will react with acid to produce HCN gas which is EXTREMELY TOXIC. Use the reagent only in the hood.

3) Follow directions carefully for the disposal of samples and unused reagents due to the nature of the solutions that are being used in this lab! Errors could prove to be deadly!!

4) All disposable glassware should be washed and rinsed as indicated prior to disposal in the white broken glass buckets in the lab.

5) Bleach solutions will be used to neutralize samples containing blood products and cyanide solutions. Be careful not to get the bleach on your clothing

The blood you will be using to perform the various tests is blood from either a sheep, cow, or goat. Because of safety and liability issues, you are not allowed to take a sample of your own blood. Although many of you are trained in the proper protocol for handling bodily fluids, and would take necessary medical precautions, not everyone in the class is trained, so we CANNOT use human blood. The animal blood is similar enough to human blood that it is sufficient for our uses. The blood has been defibrinated prior to shipment. This means that the fibrin, a protein which causes clotting of blood has been removed. This will allow you to work with the blood without it clotting. The blood should be kept cool in a cold water bath to help preserve it. It will also be necessary to hemolyze (or burst) the blood cells in the blood sample in order to release the hemoglobin molecules into the solution where they can react with the various reagents. We do this by adding a small amount of blood to a relatively large quantity of pure H2O. Since the concentration of salts inside the blood cells is higher, water will flow in through the membrane of the blood cell to try to equalize the concentration and eventually cause the cell to burst, releasing the hemoglobin into solution. Spectrophotometric Determination of Concentration of Hemoglobin You will calculate the amount of hemoglobin in a sample of animal blood (either from a sheep, cow, or goat), and do a number of tests on it. A quantitative determination of hemoglobin in humans or animals provides information for the diagnosis of anemia, in which lack of sufficient hemoglobin to carry O2 causes weakness and other symptoms.

Experiment 10 10-9 We will use the brightly colored cyanide complex of the oxidized form of hemoglobin to determine the amount of hemoglobin in our sample. Spectrophotometric determination of absorbance will be used to determine the amount of hemoglobin in a sample of blood. Because standard solutions of cyanomethemoglobin are not available for animal blood, we will use a standard solution of human hemoglobin to draw our standard curve from which we will obtain our hemoglobin concentrations. Though not terribly accurate, you will only be using two points to draw your standard curve. The wavelength at which we will be measuring the absorbance is 540 nm. 1. Make sure there are no samples in the Ultraspec. Turn on the Ultraspec. Allow the instrument to go

through its initialization processes. The machine should warm-up for 10-15 minutes before taking any readings.

2. Take 3 tubes of the hemoglobin cyanide reagent located in the hood. These three tubes will serve as your blanks. The cyanide reagent contains potassium ferricyanide (K3Fe(CN)6, a complex of cyanide with Fe3+), potassium cyanide (KCN), and a preservative.

Although the cyanide solution is stable in its current form , it is VERY important for you to keep the cyanide solution away from solutions of acid-it will react with acid to give HCN gas which is EXTREMELY toxic. UNCAP THE REAGENT ONLY IN THE HOOD. 3. Label the three tubes #1, #2, and #3, but make sure that you label the tubes above the liquid level so

that the writing does not interfere with the absorbance measurements. 4. Set the wavelength by pressing the “wave” button, entering 540 by the keypad, and pressing “enter”.

Transfer the contents of tube #1 to a plastic cuvette. Place the sample in the sample compartment in location #1. Close the lid and press “set ref”. Be sure not to change the wavelength away from 540 nm.

5. Transfer the contents of the cuvette back into tube #1. Place sample #2 into the cuvette and place it in the spectrophotometer as before. Close the lid and read the output from the instrument. Repeat this procedure with sample #3. Record the absorbance of the three blank solutions in a table in your lab notebook.

6. Measure the absorbance of the four standard solutions of human MetHbCN. These are sealed so that no bodily fluids will be exchanged. Please be careful!! The concentration of the hemoglobin used to make this solution was 5, 10, 15, and 20 g/dL. Notice that this concentration is the concentration of the hemoglobin in the original blood sample, not the concentration of MetHbCN in the sample in the tube! Since you are preparing all of the samples in a similar fashion, you do not need to know what the actual concentration of cyanomethemoglobin in the tube you use for the measurements, just the original concentration of the standard. Record the absorbances of each of these samples in your notebook; make sure that you also record the appropriate concentration for each sample. You will use the values for the blank and the standards to make the standard calibration curve to determine the hemoglobin content of the unknown.

7. Put the standards back into the cold water bath. Take your three blanks to one of the hoods with the animal blood. Use rubber gloves to protect your hands. Use a micropipet to obtain 0.02 mL of one of the types of animal blood from the hood. Your Al will show you how to use the micropipette. Use of a micropipet is a very accurate method of dispensing a very small amount of liquid.

8. IN THE HOOD: Add the blood to your cyanide reagent tube #1, cap the solution, and shake vigorously for 1 minute to mix thoroughly. Repeat the procedure for tubes #2 and #3, using the SAME type of animal blood as for tube #1, and prepare the samples in the same way. When you are finished with the micropipette, remove the plastic tip, and place in the beaker in the hood next to the pipets for proper waste disposal (do not throw in the garbage!).

Experiment 10 10-10 9. Allow the tubes to stand for 1 minute after shaking to ensure that all of the hemoglobin has reacted.

Record the color of the solution in your lab notebook. 10. Measure the absorbance of the three samples of your animal blood samples as you did with the

standards. Record the absorbances in a table in your notebook. Waste Disposal: When you are finished with your absorbance samples keep the caps on the tubes and place them in the given rack in the WASTE hood. The samples will be treated with bleach to render them harmless. ABSOLUTELY UNDER NO CIRCUMSTANCES SHOULD YOU DUMP THESE TUBES INTO THE SINK!!!!!!! IF ANY ACID IS DUMPED DOWN THE SAME SINK IT WILL PRODUCE DEADLY HCN GAS! 11. Calculate average values of absorbance for the blank. Plot the average absorbance values and Hb

concentrations of the blank (A = 0) and the standards on the graph paper provided. Mark your axes clearly. You should plot absorbance on the y-axis and concentration of hemoglobin on the x-axis. Use a ruler to draw a straight line through the points. Plot the absorbance values (A) for your unknowns along the straight line on the graph, and determine the concentration of Hb in your blood samples by reading the concentration of Hb at that absorbance value. Calculate the average concentration of Hb in your sample.

12. Obtain absorbance values from two other groups in the class for the other type(s) of animal blood which you have not used. Plot these averages on your graph and determine the concentration of hemoglobin in each of these samples.

Qualitative Analysis of Hemoglobin Derivatives Preparation of Deoxyhemoglobin (Hb) 13. Prepare two test tubes containing 10 mL of distilled H2O. Add 5 drops of blood to each one. One

of these tubes will serve as a color comparison. The blood has hemoglobin mostly in the form of oxyhemoglobin (HbO2).

14. In a third test tube, dispense approximately 2 mL of Stokes' solution. Stokes' solution contains FeSO4 (ferrous sulfate) and tartaric acid. To this solution, add NH4OH dropwise until a change in color to dark green occurs. This color is due to the formation of a complex between Fe2+ and the anion of tartaric acid, tartrate.

15. Now add drops of the ammoniated Stokes' reagent from step 14 to the oxyhemoglobin solution. Watch for a distinct color change. It will probably take 10-15 drops of Stokes solution to see a noticable color change. This should have converted the oxyhemoglobin into deoxyhemoglobin. The Fe2+ in the Stokes' reagent is more easily oxidized than the Fe2+ in oxyhemoglobin, so the Stoke's reagent pulls the O2 away from the hemoglobin, and the Fe2+ of the Stokes' reagent is oxidized by the O2. The Fe2+ in the hemoglobin remains unchanged, however. Record the color of the tubes in your notebook.

Waste Disposal: Dispose of the waste in the General Waste container in the hood (NOT in the CYANIDE WASTE container).

Experiment 10 10-11 Preparation of Methemoglobin (MetHb) 16. Dispense 5 mL of distilled H2O into each of two test tubes. Add 10 drops of blood to each. One

will serve as a color control. 17. VERY CAREFULLY add 10 drops of the 10% K3Fe(CN)6 (potassium ferricyanide (Fe3+)) solution

down the side of the tube into the solution of oxyhemoglobin. Observe the dramatic color change. If you tip the tube to the side (DO NOT SHAKE) and watch VERY carefully you might be able to see bubbles coming off (try heating the solution under warm H2O if you don't see them.) Record your observations in your notebook.

Waste Disposal: Dispose of the waste into the bleach solution labeled FOR CYANIDE WASTE in the hood. Bleach renders the cyanide harmless. Rinse out the tubes with bleach into the waste container, and then rinse with distilled H2O. Be careful not to get bleach on your clothes. Reaction of Nitrite (NO2-) with Oxyhemoglobin 18. Add 2 drops of blood to 3 test tubes containing 5 mL of distilled H2O each. One will serve as a

color control. 19. To the first tube add 1 drop of the 0.5 M NaNO2 in 0.5 M pH 4.5 acetate buffer. Observe the color

change. Record your observations in your notebook. 20. To the second test tube, add 2 drops of 0.5 M ascorbic acid in 0.5 M pH 4.5 acetate buffer. Observe

any changes in the solution and record in your notebook. 21. Then add 1 drop of 0.5 M NaNO2 in 0.5 M pH 4.5 acetate buffer to tube #2. Observe the color

changes and compare tube #2 to tube #1 and the color control solution (#3). Continue to observe tube #1 and #2 for at least another 5 minutes. Do you see any more changes? Record your observations in your notebook.

Waste Disposal: Dispose of the solutions in the GENERAL WASTE container in the hood. Effect of Heavy Metal Ions on Hemoglobin. 22. Get three small test tubes. Fill one with 5 mL distilled H2O, one with 5 mL of 0.1 M NaNO3

solution and one with 5 mL 0.1 M Pb(N03)2 solution. 23. Add 5 drops of blood to each and observe carefully what happens as you add the drops. Record

your observations in your notebook. Waste Disposal: Dispose of the solutions in the GENERAL WASTE container in the hood. Methylene Blue and Electron transfer 24. In this part of the experiment you will investigate the treatment of the "Blue Fugates" as described in

the background material. What made the "Blue Fugates" blue? The doctor who investigated the problem of the Blue Fugates found an antidote in the compound methylene blue, an organic dye. You will do an experiment showing how the dye worked to cure the Fugates of their blueness. The blue form of methylene blue is the oxidized form, while the colorless form is the reduced form.

25. Weigh out 1.00 g of glucose directly into a 200 mL Erlenmeyer flask. Add 10 mL of 20% NaOH (CAUTION NaOH is corrosive!) to the glucose solution and dilute the solution to 100 mL with distilled H2O. Swirl the solution briskly to dissolve the glucose (it may take a few minutes for all of it to dissolve).

26. To this solution, add several drops of 1% methylene blue solution. Swirl until you get a homogeneous blue color. Set the flask aside for several minutes and observe. Record your observations.

27. Swirl the solution vigorously for approximately 1 minute (careful not to spill the solution). What happened? Record your observations.

Experiment 10 10-12 28. Allow the solution to stand for a minute or so. Do you see any more color changes? 29. Repeat as many times as you wish! Waste Disposal: Dispose of the solution in the GENERAL WASTE container in the hood. Bibliography. 1. L. W. Powers, Diagnostic Hematology: Clinical and Technical Principles (C. V. Mosby: St. Louis),

1989. 2. E. N. Whitney and S. R. Rolfes, Understanding Nutrition (West: Minneapolis), 1993. 3. V. Guiliano, J. P. Reick, Hemoglominometry-A Biochemistry Experiment that Utilizes Principles of

Transition Metal Chemistry, J Chem. Ed., 1987, 64, 354-355. 4. Merck Veterinarv Manual-A Handbook of Diagnosis and Therapy for the Veterinarian, 5th Edition, (Merck &

Co., Rahway, NJ), 1979. 5. A. E. Fenster, D. N. Harpp, J. A. Schwarcz, J. 0. Olanville, A Well-Known Medical Chemical

Demonstration to Illustrate an Unusual Medical Mystery, J Chem. Ed., 1988, 65,621. 6. S.F. Russo, R. B. Sorstokke, Hemoglobin, J Chem. Ed., 1973, 50, 347-350. 7. R. E. Dickerson, I. Geis, Hemoglobin: Structure, Function, Evolution, and Pathology (Benjamin

Cummings: Menlo Park, CA), 1983.

C. Discussion Questions for Your Laboratory Report Make sure that the following items are included in your laboratory report. Remember this is not an exhaustive list. Remember you are responsible for explaining all observations that are made. 1. Prepare a table of for recording spectrophotometer readings for the blanks, the standards, and the

unknowns. 2. Show the calculations necessary to determine the average Hb values for the blank, and the unknown

Hb values for the blood that you tested as well as the other blood samples available in the laboratory. 3. Record all qualitative data – color changes, bubbling, etc – that you observe throughout the course of

this experiment. Make sure that you carefully label these observations so you (and your AI) know which part of the laboratory your observations relate to.

Specific questions to address in your discussion as you explain your observations: 1 Based on your data, do you see any differences in the concentration of hemoglobin in the three types

of animal blood used? If so, why might some animals have more hemoglobin than others? How does this compare to the values listed in the table in the introduction to this laboratory? Why might your values be different from those presented in table 2?

2. What reaction occurs when Stoke's reagent is added to the oxyhemoglobin solution? 3. If the Fe2+ in your hemoglobin is oxidized to Fe3+ in this reaction, what element is being reduced?

Write a simple equation for this reduction. 4. Why might you be able to see bubbles coming off when you oxidize hemoglobin to methemoglobin

(i.e. what gas could be produced)? Why would it be bad if this oxidized form of hemoglobin was the only one present in the body?

5. Explain the change in color that you saw when you added only NaNO2 to your solution of oxyhemoglobin (What is the new product?)

6. What is the product of the reaction of NaNO2 and ascorbic acid with oxyhemoglobin? 7. Why does the meat industry add nitrites to meats? 8. What happens to the hemoglobin when it is exposed to Pb?

Experiment 10 10-13 9. What compound caused the "Blue Fugates" to look blue? 10. What is the difference between the compound listed above and normal hemoglobin? 11. What color was the species you made in the Blue Fugates experiment? 12. What color is methemoglobin when viewed through the skin? 13. What happened to your blue solution upon standing? 14. Why might glucose serve the same purpose as the pyridine nucleotides in the bodies of the Blue

Fugates (Hint: re-read the section on the Blue Fugates and go to your C102 text to determine what type of sugar is glucose?)?

15. What happened to your solution upon swirling? 16. What do you think caused this? (Hint the blue form is oxidized, the colorless form is reduced). What

is performing the oxidation? 17. When methylene blue pills were given to the Blue Fugates, what caused the oxidation of the

methylene blue back to the blue form?

Experiment 10 10-14

Experiment 9 Prelab 9-1 Name: Section:

Blood and Hemoglobin 1. What are the two major parts of the protein hemoglobin? Define each of these? 2. What is the function of hemoglobin? 3. What is meant by the term methemoglobin? 4. Draw the Lewis dot structures of carbon monoxide and oxygen gas. Why might these two molecules

act similarly in the body? Why is this a problem? 5. How will you dispose of the cyanomethemoglobin solutions that you measure the absorbance of? 6. How will you dispose of the methemoglobin solution that you make with ferricyanide? 7. How will you dispose of all of your other waste solutions?