An Undergraduate Laboratory Experiment in Bioinorganic Chemistry

May 6, 2011 - Suchol Savagatrup , Vera Schroeder , Xin He , Sibo Lin , Maggie He , Omar Yassine , Khaled N. Salama , Xi-Xiang Zhang , Timothy M. Swage...
4 downloads 0 Views 883KB Size

An Undergraduate Laboratory Experiment in Bioinorganic Chemistry: Ligation States of Myoglobin James A. Bailey* Barber School of Arts and Sciences Unit 3 - Chemistry, University of British Columbia—Okanagan, Kelowna BC V1V 1V7, Canada

bS Supporting Information ABSTRACT: Although there are numerous inorganic model systems that are readily presented as undergraduate laboratory experiments in bioinorganic chemistry, there are few examples that explore the inorganic chemistry of actual biological molecules. We present a laboratory experiment using the oxygen-binding protein myoglobin that can be easily incorporated into an undergraduate laboratory course. This highly intuitive experiment in which ligand-binding reactions of carbon monoxide and dioxygen are monitored using scanning visible absorption spectroscopy provides the opportunity to investigate the relative binding strengths of O2 and CO to the reduced iron porphyrin center. This process represents an important first step in the mechanism of respiration and offers a convenient entry point into several interesting topics for discussion including, for example, the role of metals in biology; ligand binding and redox states in metalloporphyrins; the application of spectroscopic probes to the study of kinetics and mechanism; protein dynamics; and structurefunction relationships. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Inorganic Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioinorganic Chemistry, Biophysical Chemistry, Proteins/Peptides, UVVis Spectroscopy


he inorganic chemistry of biological molecules is a fascinating topic and there are many systems that are sufficiently well understood such that a classroom course can be easily filled with studies of both biological systems and the corresponding synthetic model systems. Unfortunately, for the accompanying laboratory course, whereas many of the numerous inorganic model systems are readily adapted to an undergraduate lab,14 there are few similarly manageable examples that explore the inorganic chemistry of an actual biological molecule.5 A simple and intuitive study of the ligation states of myoglobin (Mb) is presented that can be included in an undergraduate laboratory with very little specialized glassware or equipment required. The entire experiment is based on qualitative observation using UVvis absorption spectroscopy and has been successfully implemented in our third-year undergraduate bioinorganic chemistry course. Biological transformations are often facilitated by the presence of a prosthetic group containing a functionally requisite metal atom intimately associated with a protein or enzyme. Although the reactivity of these systems is often primarily centered at the metal, the protein framework is critical for both fine-tuning of the reactivity and regulation of substrate approach to the metal active site. An excellent example is found in myoglobin, an oxygen-binding protein found in muscle tissue that is similar to hemoglobin in many respects. The structures of hemoglobin and Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

myoglobin share some important features including the metal cofactor and basic tertiary arrangement of the surrounding protein. To bind dioxygen, the iron center must first be reduced from the þ3 (metmyoglobin, metMb) to the þ2 oxidation state. Reduced Fe(II) porphyrins normally bind π-acceptor ligands such as CO very strongly. To steer binding toward oxygen in preference to other ligands, the binding pocket contains an additional histidine amino acid side chain (the distal histidine) that provides steric bulk to destabilize a linearly bound ligand (e.g., CO) and that can take part in hydrogen bonding to an appropriately positioned negatively charged atom (e.g., a δ oxygen atom); hence, the stabilization of the O2 ligand (Figure 1). For myoglobin, these two effects provide nearly 1000-fold increase in relative binding affinity for O2.6,7 Unfortunately, even with this tuning of the binding pocket, CO is still more strongly bound to myoglobin (and hemoglobin) than is O2 (KO2 = 0.92  106 L mol1; KCO = 14.5  106 L mol1).8 Although CO has an important physiological role,9 it is also highly toxic owing to this high affinity; CO is displaced only very slowly by an excess of oxygen. In conjunction with the reactivity presented in this experimental procedure, myoglobin presents an Published: May 06, 2011 995 | J. Chem. Educ. 2011, 88, 995–998

Journal of Chemical Education


Figure 1. Binding pocket of myoglobin showing (A) stabilization of bound O2 by interaction with the distal histidine and (B) steric destabilization of CO ligand. The CO ligand is noticeably tipped off the preferred 90° bonding angle for N(porph)-Fe-CO. The tip in this drawing is slightly exaggerated; the actual tip is 9° off normal.10

Figure 2. Absorbance spectra of myoglobin/Na2S2O4 solution following successive additions of oxygen from air. The absorbance at 320 nm due to excess dithionite decreases with each successive 1 mL aliquot of air bubbled through the cuvette indicating that the unreacted dithionite is being consumed before ultimate formation of MbO2.

excellent opportunity to explore the structural aspects that are relevant to the tuning of the ligand-binding pocket: the crystal structures of MbCO (1A6G) and MbO2 (1A6M)10 are both available in the Protein Data Bank. The transformations between redox and ligation states of Mb can be readily followed using UVvis spectroscopy. The intense coloration of heme proteins is due principally to the ππ* transitions associated with the porphyrin ring.11 As seen in Figure S1 (in the Supporting Information), for myoglobin there is an intense UV absorption that occurs between 400 and 450 nm (the Soret or B band) and a less intense series of bands in the visible region near 550 nm (the Q bands). The peak maxima of the B and Q bands shift depending on oxidation state and ligation state of the metal center, giving a convenient and diagnostic spectral signature to follow.

following small additions of air (Figure 2). When the excess reducing agent is consumed, O2 will then immediately bind to the reduced heme causing the Soret band to shift from 435 to 419 nm. In the third step, the ligand-exchange reaction can be observed by adding CO gas to the cuvette containing the oxymyoglobin. As shown in the Supporting Information, the reaction ratea is approximately equal to the MbO2 dissociation rate (k0 O2 = 12 s1)17 so MbCO forms within a few seconds of CO introduction, and once formed, the MbCO dissociation is sufficiently slow (k0 CO = 0.035 s1)8 that the exchange goes quickly to completion. The CO gas is conveniently prepared in small, easily manageable quantities by reaction of formic acid or sodium formate with concentrated sulfuric acid in an N2-purged flask fitted with a septum cap. Using about 12 mL each of formic acid and sulfuric acid, sufficient quantity of CO gas is formed within a minute or two. The easiest method is to collect the CO gas with a syringe and then bubble it through the oxymyoglobin solution in the cuvette. This exchange occurs very quickly within the first 1 mL of CO gas addition.b This can be qualitatively interpreted as a fast O2 off-rate for comparison to the CO off-rate described below. With the ligand exchange, the Soret maximum shows a small shift from 419 to 423 nm and a significant narrowing and increase in peak intensity. This change is quite obvious if the spectra are overlaid as they are collected. An alternate way to form the MbCO (and slightly more rigorous in terms of concentrations) is by manually mixing CO-containing bufferc with a solution of oxymyoglobin; in this case, there is complete conversion to carbon monoxymyoglobin within the mixing time, giving an absorbance maximum at 423 nm.

’ EXPERIMENT Part 1: MbO2 Reaction with CO

The entire experiment can be carried out in a standard UVvis 3.5 mL cuvette. The myoglobin concentration should be around 10 μM but is not critical: a metmyoglobin concentration that gives an absorbance at 409 nm between 1.0 and 1.2 in a 1 cm cuvette is adequate. In the first step, sodium dithionite (Na2S2O4) is used as a reducing agent12 that serves a dual purpose: it removes dissolved dioxygen by reducing it to water13 and reduces the Fe(III) in metMb to Fe(II),1416 which results in a large shift in the Soret maximum from 409 to 435 nm. (See Scheme S1 in the Supporting Information for half-reactions and reduction potentials.) There is no need to provide an inert atmosphere in the headspace of the cuvette because oxygen diffusion is sufficiently slow and the presence of excess dithionite ensures that no oxymyoglobin forms prematurely. These reactions will lower the pH and potentially denature and precipitate the myoglobin protein; therefore, a buffered solution is necessary (0.1 M phosphate; pH ∼7). In the second step, dioxygen (from air) is bubbled through the reduced myoglobin solution in the cuvette. Dioxygen will react with the excess S2O42 and will not bind with Mb until S2O42 is consumed; therefore, the excess dithionite must be removed first by reaction with O2. This process is conveniently monitored by observing the loss of the dithionite absorbance at 320 nm

Part 2: MbCO Reaction with O2

The second half of this experiment is composed of a parallel procedure in which the carbon monoxymyoglobin is prepared first and then the CO is displaced by oxygen from air. By comparing the ease with which the ligand exchange occurs, a qualitative measure of the relative binding strengths of the two ligands is readily apparent. Only minor changes from the first procedure are required. In particular, after preparation of reduced myoglobin, the excess dithionite does not need to be 996 |J. Chem. Educ. 2011, 88, 995–998

Journal of Chemical Education


the protein scaffold in metalloproteins, equilibrium binding kinetics and thermodynamics, and the protein structure function relationship.



Supporting Information Instructor notes, detailed experimental procedures, and a student handout. This material is available via the Internet at

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

Figure 3. Successive additions of air to MbCO solution (15 μM). Inset shows expanded view of Soret band with well-defined isosbestic point.

’ ADDITIONAL NOTE a Different reaction rates will be observed depending on the source of myoglobin. Myoglobin from equine skeletal muscle was used for all experiments reported here. A complete set of rate constants is given in the Supporting Information. b

The actual volume of CO gas required depends on the quantity of gas produced in the gas-generating apparatus.

titrated as it will not react with CO. MbCO forms quickly with the first small addition of CO gas to reduced myoglobin; subsequent slow bubbling of air will first remove the excess dithionite as before and then begin to react with MbCO. The exchange reaction is now significantly slower, limited by the dissociation of MbCO (k0 CO = 0.035 s1)8 and requiring as much as 30 mL of air to displace the CO ligand. During the course of the ligand exchange, the Soret maximum will slowly shift from about 423 to 419 nm with simultaneous broadening and decrease in intensity (Figure 3). This process is now slow enough that a manual mixing method can be used to collect (nearly) complete kinetic traces. By mixing aerated buffer with myoglobin solution (50 μM) in a 4:1 ratio, quickly placing it in the spectrometer, and following the reaction using either single wavelength (e.g., 423 nm) or full spectral scans, clean first-order kinetic profiles are easily obtained as shown in Figure S2 (in the Supporting Information). The reaction is complete in about 23 min and gives a rate constant that is consistent with the literature value.8

’ HAZARDS Carbon monoxide is a toxic gas. The gas generator apparatus should be set up in a fume hood and all manipulations and additions to the myoglobin solution should also be carried out in the fume hood. Sulfuric and formic acids are highly corrosive; use caution and handle in the fume hood using gloves and eye protection. Formic acid is a combustible liquid and vapor. Sodium dithionite is a flammable solid and may ignite with moisture and air. It can cause irritation to the skin, eyes, and respiratory tract.


If a CO-gas cylinder is employed, a saturated solution can be obtained relatively easily by bubbling CO gas through the deaerated buffer solution for a few minutes: under 1 atm pressure at 25 °C, [CO] = 1 mM. The concentration of dissolved CO is difficult to control when using the laboratory preparation of CO gas; however, we have found that bubbling 1520 mL of the gas obtained from the gas generator and using a 4:1 ratio of CO buffer to myoglobin solution (50 μM) easily provides sufficient [CO] to satisfy the requirements necessary for fast ligand exchange. See the Supporting Information for more details.


During review it was noted by one of the referees that this procedure, in addition to its use as a stand-alone experiment in bioinganic chemistry, could be incorporated into a biochemistry laboratory module comprised of a set of experiments many of which are available in the current literature. For example, a module could include: extraction and purification of myoglobin from hamburger,18 myoglobin unfolding and stability by any of a number of different techniques,1921 and this ligation study.

’ REFERENCES (1) Donlin, M. J.; Frey, R. F.; Putnam, C.; Proctor, J. K.; Bashkin, J. K. J. Chem. Educ. 1998, 75, 437–441. (2) McQuate, R. S. J. Chem. Educ. 1977, 54, 645–648. (3) McQuate, R. S.; Reardon, J. E. J. Chem. Educ. 1978, 55, 607–609. (4) Williams, K. R.; Adhyaru, B. J. Chem. Educ. 2004, 81, 1045–1047. (5) Battin, E. E.; Lawhon, A.; Brumaghim, J. L.; Hamilton, D. H. J. Chem. Educ. 2009, 86, 969–972. (6) Cao, W.; Ye, X.; Georgiev, G. Y.; Berezhna, S.; Sjodin, T.; Demidov, A. A.; Wang, W.; Sage, J. T.; Champion, P. M. Biochemistry 2004, 43, 7017–7027. (7) Jameson, G. B.; Ibers, J. A. Dioxygen Carriers. In Biological Inorganic Chemistry: Structure and Reactivity; Bertini, I., Gray, H. B., Stiefel, E. I., Valentine, J. S., Eds.; University Science Books: Sausalito, CA, 2007. (8) Wan, L.; Twitchett, M. B.; Eltis, L. D.; Mauk, G.; Smith, M. Proc. Nat. Acad. Sci. U.S.A. 1998, 95, 12825–12831.

’ CONCLUSION The ligand-exchange reaction described here presents a simple and intuitive experiment that can easily be performed by an undergraduate chemistry student.d This experiment lends itself to a broad range of discussion topics such as electronic spectroscopy of metalloporphyrins, the role of 997 |J. Chem. Educ. 2011, 88, 995–998

Journal of Chemical Education


(9) Verma, A.; Hirsch, D. J.; Glatt, C. E.; Ronnett, G. V.; Snyder, S. H. Science 1993, 259, 381–384. (10) Vojtechovsky, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Biophys. J. 1999, 77, 2153–2174. (11) Gouterman, M. J. Mol. Spectrosc. 1961, 6, 138–163. (12) Mayhew, S. G. Eur. J. Biochem. 1978, 85, 535–547. (13) Harris, D. C. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010; p 298. (14) Cox, R. P.; Hollaway, M. R. Eur. J. Biochem. 1977, 74, 575–587. (15) Liu, A.; Wei, M.; Honma, I.; Zhou, H. Anal. Chem. 2005, 77, 8068–8074. (16) Taylor, J. F.; Morgan, V. E. J. Biol. Chem. 1942, 144, 15–20. (17) Springer, B. A.; Egeberg, K. D.; Sligar, S. G.; Rohlfs, R. J.; Mathews, A. J.; Olson, J. S. J. Biol. Chem. 1989, 264, 3057–3060. (18) Bylkas, S. A.; Andersson, L. A. J. Chem. Educ. 1997, 74, 426–430. (19) Mehl, A. F.; Crawford, M. A.; Zhang, L. J. Chem. Educ. 2009, 86, 600–602. (20) Sykes, P. A.; Shiue, H. C.; Walker, J. R.; Bateman, R. C. J. Chem. Educ. 1999, 76, 1283–1284. (21) Schuh, M. D. J. Chem. Educ. 1988, 65, 740–741.

998 |J. Chem. Educ. 2011, 88, 995–998