Myoglobins

Myoglobin (Mb) is a hemoprotein capable of reversible fixation of Oj in muscles of vertebrates and invertebrates. Cotton et al. have thus compared the oxidation and spin-state markers of RR bands in solution and at a Ag electrode. From SERRS spectra of adsorbed Mb at two adsorption potentials, —0.6 V and —0.2 V vs. SCE, 

Soluble proteins make up 25-30% of the total protein in muscle tissue. They consist of ca. 50 components, mostly enzymes and myoglobin (cf. Table 12.5). The high viscosity of the sarcoplasm is derived from a high concentration of solubilized proteins, which can amount to 20-30%. The glycolytic enzymes are bound to the myofibrillar proteins in vivo. 

Sarcoplasm contains most of the enzymes needed to support the glycolytic pathway and the pentosephosphate cycle. Glyceraldehyde-3-phosphate dehydrogenase can make up more than 20% of the total soluble protein. A series 

Muscle tissue dry matter contains an average of 1% of the purple-red pigment myoglobin. However, the amounts in white and red meat vary considerably. 

For hemoglobin, n 2.8 (sigmoidal saturation curve), and for myoglobin, n = 1 (hyperbolic saturation curve). The efficiency of O2 transfer from hemoglobin to myoglobin is further enhanced by a decrease in pH since oxygen binding is pH-dependent (the Bohr effect). 

Heme devoid of globin (free heme, Fe +-protoporphyrin) does not form the O2-adduct, but oxidizes rapidly to hemin (Fe +-protoporphyrin). A prerequisite for reversible O2 binding is the presence of an effective donor ligand on the iron s axial site, which is bound by formation of a quadratic-pyramidal complex. The imidazole 

Another form of cross-linking is encountered when Mb is reacted with H2O2 under mildly acidic conditions. Formation of the so-called haem-to-protein cross-linked myoglobin (Mb-H), which is reported to possess potent per-oxidative activity, involves the generation of an intramolecular covalent bond between the porphyrin and protein moieties of myoglobin. Mb-H is excreted in the urine of patients suffering renal failure as a result of rhabdomyolysis (muscle 

Myoglobin can also cross-link to other proteins, including LPO and albumin. Cross-linking to albumin is believed to involve oxidation of the protein by Mb-Fe(III)/H202, resulting in the generation of an albumin-centred radical that has been observed at 77 K. In a recent study, a broad, featureless signal was observed at room temperature.  

The trapping of the ty rosyl-103 radical in Mb by DMPO has been exploited in the development of an immunoassay by Detweiler and colleagues.58 Antibodies were raised against a derivative of DMPO and then used to detect the Mb adduct in rat heart supernatant treated with H202. The technique has also been used to detect a protein-tyrosyl radical from haemoglobin in red blood cells exposed to H202.59 

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There has been extensive work done on myoglobin, haemoglobin, Cytocln-ome-c, rhodopsin and bacteriorhodopsin. In fact, there are literally hundreds of articles on each of the above subjects. Flere we will consider haemoglobin [12]. The first tliree of these examples are based on the protohaeme unit, shown in figure Bl.2.10. 

Figure Bl.2.10. Structure of the protohaeme unit found in haemoglobin and myoglobin.

Figure Bl.2.11. Biologically active centre in myoglobin or one of the subunits of haemoglobin. The bound CO molecule as well as the proximal and distal histidines are shown m addition to the protohaeme unit. From Rousseau D L and Friedman J M 1988 Biological Applications of Raman Spectroscopy vol 3, ed T G Spiro (New York Wiley). Reprinted by pennission of John Wiley and Sons Inc.

Asher S A and Chi Z H 1998 UV resonance Raman studies of protein folding in myoglobin and other proteins Biophys. 

Owrutsky J C, Li M, Locke B and Hochstrasser R M 1995 Vibrational relaxation of the CO stretch vibration in hemoglobin-CO, myoglobin-CO, and protoheme-CO J. Rhys. Chem. 99 4842-6... 

Genberg L, Richard L, McLendon G and Miller R J D 1991 Direct observation of global protein motion in hemoglobin and myoglobin on picosecond time scales Science 251 1051-6... 

Rella C W, Rector K D, Kwok A, Hill J R, Schwettman H A, DIott D D and Fayer M D 1996 Vibrational echo studies of myoglobin-CO J. Phys. Chem. 100 15 620-29... 

Elber R and Karplus M 1987 A method for determining reaction paths in large molecules application to myoglobin Chem. Phys. Lett. 139 375... 

Lewis J W, Tilton R F, Einterz C M, Milder S J, Kuntz I D and Kliger D S 1985 New technique for measuring circular dichroism changes on a nanosecond time scale. Application to (carbonmonoxy)myoglobin and (carbonmonoxy)hemoglobin J. Rhys. Chem. 89 289-94... 

Hill J R ef a/1994 Vibrational dynamios of oarbon monoxide at the aotive site of myoglobin piooseoond infrared free-eleotron laser pump-probe experiments J. Phys. Chem. 98 11213-19... 

We assume in the following that the ligand is bound in a binding pocket of depth 6 —a = 7 A involving a potential barrier AU = 25 kcal/mol, similar to that of streptavidin (Chilcotti et al., 1995). We also assume that the diffusion coefficient of the ligand is similar to the diffusion coefficient of the heme group in myoglobin (Z) = 1 A /ns) as determined from Mofibauer spectra (Nadler and Schulten, 1984). 

The problems that occur when one tries to estimate affinity in terms of component terms do not arise when perturbation methods are used with simulations in order to compute potentials of mean force or free energies for molecular transformations simulations use a simple physical force field and thereby implicitly include all component terms discussed earlier. We have used the molecular transformation approach to compute binding affinities from these first principles [14]. The basic approach had been introduced in early work, in which we studied the affinity of xenon for myoglobin [11]. The procedure was to gradually decrease the interactions between xenon atom and protein, and compute the free energy change by standard perturbation methods, cf. (10). An (issential component is to impose a restraint on the... 

Hermans, J., Subramaniam, S. The free energy of xenon binding to myoglobin from molecular dynamics simulation. Isr. J. Chem. 27 (1986) 225-227... 

Elber R and M Karplus 1987. A Method for Determining Reaction Paths in Large Molecules Application to Myoglobin. Chemical Physics Letters 139 375-380. 

Fiber R and M Karplus 1990. Enhanced Sampling in Molecular Dynamics Use of the Time-Dependent Hartree Approximation for a Simulation of Carbon Monoxide Diffusion through Myoglobin. Journal of the American Chemical Society 112 9161-9175. 

For their work on myoglobin and hemoglobin respec tively John C Kendrew and Max F Perutz were awarded the 1962 Nobel Prize in chemistry... 

Most potential energy surfaces are extremely complex. Fiber and Karplus analyzed a 300 psec molecular dynamics trajectory of the protein myoglobin. They estimate that 2000 thermally accessible minima exist near the native protein structure. The total number of conformations is even larger. Dill derived a formula to calculate the upper bound of thermally accessible conformations in a protein. Using this formula, a protein of 150 residues (the approx-... 

Fiber, R. Karplus, M. Multiple conformational states of proteins a molecular dynamics analysis of myoglobin. Science 235 318-321, 1987. 

Quasi-molecular ions, [M + nH], from a protein (myoglobin) of molecnlar mass 16,951.5 Da. In this case, n ranges from 21 (giving a measured mass of 808.221) to 12 (corresponding to a measured mass of 1413.631). The peaks with measured masses in between these correspond to the other values of n between 12 and 21. By taking snccessive pairs of measnred masses, the relative molecular mass of the myoglobin can be calculated very accurately, as shown in Figure 8.4. 

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

A sample of the protein, horse heart myoglobin, was dissolved in acidified aqueous acetonitrile (1% formic acid in HjO/CHjCN, 1 1 v/v) at a concentration of 20 pmol/1. This sample was injected into a flow of the same solvent passing at 5 pl/min into the electrospray source to give the mass spectrum of protonated molecular ions [M + nH] shown in (a). The measured ra/z values are given in the table (b), along with the number of protons (charges n) associated with each. The mean relative molecular mass (RMM) is 16,951,09 0.3 Da. Finally, the transformed spectrum, corresponding to the true relative molecular mass, is shown in (c) the observed value is close to that calculated (16,951.4), an error of only 0.002%. 

The color of red meat depends on oxygen. The color of the meat pigment myoglobin is purple. The bright red color of the fresh-as-cut meat is from oxymyoglobin. To preserve red meat, the objectives are to retard spoilage and weight loss, and to deUver red color at the consumer level. 

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