Exp t 114 Microburger Biochemistry Isolation of Myoglobin from Hamburger Adapted by Don Miller and R Minard from Dylkas Sheri A Anderson Laura A J Chem Ed 1997 74 426 430 Rev 10 16 01 Introduction Mammalian myoglobin Mb is a monomeric oxygen binding protein found in cardiac and skeletal muscles 1 It binds more tightly to oxygen than hemoglobin so it can effectively extract oxygen from the blood Myoglobin the brownish red pigment in the dark meat of poultry fish and beef is often found in muscles that make use of a steady supply of energy 2 The protein has a single heme group the part of the protein not made up of amino acids named iron protoporphyrin IX that is ligated a lewis base attached to a metal atom in a complex to the protein by histidine residue His 93 3 C H2 H H C H2 N N N N H H O O 3 Fe 2 Fe N N N H 2C H O H 2C N Met myoglobin Oxy myoglobin The experiment demonstrates the two common and stable forms of the protein Fe 3 H2O Mb Met Mb and Fe 2 O2 Mb Oxy Mb In living organisms Oxy Mb and deoxy Mb are the two most common forms However in a nonliving system Oxy Mb is slowly converted to Met Mb as the heme bound O2 molecule is released and an active site H2O molecule is bound This redox activity can be recreated in the lab by oxidizing Fe 2 ferrous heme to Fe 3 ferric form and reducing Fe 3 to Fe 2 The ferric form Met Mb will be yellow to yellowish brown and the ferrous form Oxy Mb will be a bright red color enabling a direct correlation of bright red Oxy Mb to fresh meat and the yellow brownish color to old meat 1 2 Fe 3 Fe O2 Mb e 3 ox agent Fe H 2O Mb red agent e H2O Mb 2 Fe O 2 Mb Prelaboratory Exercise In the study of redox reactions like the one above it is important to note the many other types of redox reactions that are possible One such example is the reduction of potassium permanganate by sodium dithionite If you were to place 2 mL of a dilute aqueous solution of potassium permanganate KMnO4 in a small beaker with water you would notice the purple color of the potassium permanganate If you then added a reducing agent such as sodium dithionite sodium hydrosulfite Na2 S2 O4 dropwise to the beaker you would observe a color change from purple to pale pink as the permanganate ion is reduced to Mn2 and the dithionite is oxidized to sulfite SO3 2 Write and balance the redox equation for this chemical reaction 1 Dylkas Sheri A Anderson Laura A J Chem Ed 1997 74 426 430 F 97 2Campbell Neil A Biology The Benjamin Cummings Publishing 1993 1043 3Campbell Neil A Biology The Benjamin Cummings Publishing 1993 184 4 Lenga Robert E Votoupal Kristine L Sigma Aldrich Library of Regulatory and Safety Data 1993 3 3519 E Exp t 114 Calculate the weight of potassium phosphate monobasic KH2 PO4 FW 136 and potassium phosphate dibasic K2 HPO4 FW 174 required to prepare 20 mL of a pH 6 8 phosphate buffer solution that is 20 mM in potassium phosphate monobasic KH2 PO4 and 20mM in potassium phosphate dibasic K2 HPO4 Cautions The isolation of myogolbin requires the use of a bench top centrifuge Be sure to balance the centrifuge with two tubes of equal weight when operating or damage can occur to the centrifuge and the centrifuge tube may shatter Potassium ferricyanide is a harmful solid that is incompatible with strong acids and strong oxidizing agents4 Isolation of myoglobin from hamburger Prepare 20 mL of a pH 6 8 phosphate buffer solution that is 20 mM in potassium phosphate monobasic KH2 PO4 and 20mM in potassium phosphate dibasic K2 HPO4 Prepare a 4g microburger and place it in a 10 mL plastic centrifuge tube which can be obtained from a rack next to the Beckman centrifuge in lab 215 Add 5 mL of the phosphate buffer to the hamburger in the centrifuge tube The burger needs to be stirred with a glass stirring rod for one min in order to break open cells and release Mb Over stirring will cause a release of fats and nucleic acids in the sample Make sure the centrifuge is balanced by placing another plastic centrifuge tube filled to the same level as your burger tube at a position 180 opposite the burger tube The burger buffer solution is centrifuged for 5 min at 10 000 rpm according to the instructions posted on the wall above the centrifuge Isolation and Purification A red layer at the top and a meat pellet layer at the bottom are observed The red layer containing the Mb can be carefully removed by a pipet If the solution is turbid or cloudy it should be filtered through a filter pipet containing cotton and 1 2 of Celite filter aid A dilute solution of the Mb 1 0mL with 3 0mL of initial buffer is analyzed by electronic absorption UV Vis spectroscopy The remaining Mb is divided in half One half of the sample can be oxidized by mixing with 15 crystals 10 30 mg of potassium ferricyanide A yellow brown color will be present The other half is reduced by mixing with approximately 15 crystals 10 30 mg of sodium dithionite A bright red color will be present Both samples should sit for five minutes and then the absorbance spectra should be recorded diluting with buffer as required to get an absorbance below 2 AU in the range 300 to 800 nm The absorbance should be recorded for the oxidized reduced and initial Mb solutions Cleaning up Clean the plastic centrifuge tubes with soap and water rinse with distilled water NOT ACETONE and place upside down in the rack by the Beckman centrifuge All reagents used should be rinsed down the sink with a large amount of water Extra hamburger should be disposed of by wrapping in a paper towel and placing this in the waste bin Analysis The products should be analyzed by a Hewlett Packard 5842 Diode Array UV Visible spectrophotometer Final Report Make a table of UV Vis spectral peak absorbances or lmax Compare lmax for the initial solution with lmax for the oxidized and reduced solutions Describe any differences similarities in absorptions between the oxidized form or the reduced form and the initial Mb solution 1 What peaks are similar between the initial Mb solution and the oxidized reduced solutions 2 Why does meat brown as it ages Synthetic Experiment PreLab Grading Sheet Name s TA Date PreLab For Exp t 114 Microburger Biochemistry Isolation of Myoglobin From Hamburger Possible Points Date Name Desk Experiment Title abbreviated after 1st pg Section TA Name 4 Summary 14 Goals 8 Myoglobin redox reaction with structures extraction diagram 14 Completeness of Chemical Data Table s most information for starting material hamburger is not applicable N A 14 Spectral