r2010 American Chemical Society and Division of Chemical Education, Inc. _ pubs.acs.org/jchemeduc _ Vol. 88 No. 2 February 2011 _ Journal of Chemical Education 223 10.1021/ed100585t Published on Web 11/17/2010 In the Laboratory What Is the True Color of Fresh Meat? A Biophysical Undergraduate Laboratory Experiment Investigating the Effects of Ligand Binding on Myoglobin Using Optical, EPR, and NMR Spectroscopy Kimberly Linenberger, Stacey Lowery Bretz, Michael W. Crowder, Robert McCarrick, Gary A. Lorigan,* and David L. Tierney* Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States *garylorigan@muohio.edu ( G.A.L.) and tiernedl@muohio.edu ( D.L.T.). The new ACS Committee on Professional Training guide- lines call for more integrated upper-level undergraduate labora- tories (1). However, few such experiments have been described in the literature. A review of approximately 250 modern physical chemistry experiments, covering both laboratory instrumenta- tion and current research topics, found only seven could be con- sidered integrated (2). A comparison of the experimental tech- niques used in chemistry research and those published for use in undergraduate laboratories, presented in this Journal, found several obvious omissions (3). One underrepresented technique is electron paramagnetic resonance (EPR) spectroscopy. Only four experiments that use EPR spectroscopy have appeared in this Journal since 2000 (4-7). The experiment discussed below combines modern spectroscopic techniques (EPR, NMR, and optical spectroscopy), set in the context of better understanding meat packaging practices in the United States. Controversy has been sparked with the revelation that red meat is often packaged in an oxygen free, carbon monoxide rich environment (8-10). The brown color associated with the aging process is chiefly the result of air oxidation of myoglobin (Mb), upon prolonged exposure to oxygen. Packaging in a CO atmos- phere preserves the pink color associated with freshness, making the meat appear fresher than it actually is. In this experiment, students investigate this issue by examining the effect of ex- changing the distal ligand of Mb on its optical and magnetic properties. Myoglobin is a single chain, 154 amino acid poly- peptide (17 kDa) that folds into eight R-helices (11); it contains a single heme, coordinated proximally by a histidine side chain and distally by a water molecule. In its reduced Fe(II) form, Mb, which is ubiquitous in muscle tissue, binds and stores oxygen in muscle for use under conditions of stress and normal cellular metabolism. Oxidation of the heme to Fe(III) eliminates O 2 binding and is responsible for the color change that is observed as red meat ages. The color change depends on both the oxidation state of the iron and the identity of the distal ligand. When beef is first cut, the meat is a purple color due to the prevalence of reduced myoglobin. Upon exposure to air, the Fe(II) binds oxygen, forming oxymyoglobin, which is responsible for the bright pink color often associated with freshness. However, upon prolonged exposure to oxygen, the iron oxidizes to form met- myoglobin (metMb), which is brown (12-14). The addition of CO converts high-spin aquometMb to the low-spin metMbCO complex, which is close in color to the pink observed in oxy- myoglobin. Addition of azide to aquometMb results in a similar change in spin state and therefore color. Myoglobin has been the focus of multiple laboratory investi- gations described in this Journal (15-21), including a detailed optical titration of metMb with azide (16). However, the present experiment is the first to incorporate EPR and paramagnetic NMR spectroscopy, for any system. Guided by UV-visible spectroscopy, paramagnetic resonance is used to examine the effect of replacing the coordinated water molecule in resting metMb with the azide anion, which converts the ferric heme from high to low spin. A similar conversion takes place on formation of the CO adduct, making the connection to the meat packing process. The laboratory consists of (i) UV-visible, (ii) continuous wave (CW) EPR, and (iii) NMR spectroscopy of metMb, with (metMbN 3 ) and without (aquometMb) added azide. Given pre-laboratory instruction introducing EPR and paramagnetic NMR spectroscopy, the experiments can be com- pleted in two 3-4 h laboratory periods with students working in pairs. This biophysical chemistry experiment is appropriate for an upper-level undergraduate laboratory course. Experimental Procedure Samples of Mb were prepared for spectroscopy (22) by dissolving commercially available, lyophilized horse skeletal muscle Mb in 0.2 M phosphate buffer at pH 7. Mb was fully oxidized to metMb by the addition of a 2-fold molar excess of K 3 Fe(CN) 6 , and subsequent dialysis, according to published procedures (16). NMR samples were prepared using buffer made from 99.8% D 2 O and adding K 3 Fe(CN) 6 directly. The metMbN 3 complex was subsequently formed by adding a 2-fold molar excess of NaN 3 . UV-Visible Spectroscopy Optical spectra were collected for both 10 and 80 μM samples of aquometMb and metMbN 3 using an Agilent model 8435 diode array spectrophotometer. Typical spectra are shown in Figure 1A. Students were asked to determine transition energies in both reciprocal centimeter (cm -1 ) and joules, from