Myoglobin titration

In a recent study, Hermans (1962) has indicated that only two of the three tyrosyl phenolic groups of myoglobin titrate below pH 13, this result being obtained for both whale and horse myoglobins. 

The kinetics of reactions of NO with ferri- and ferro-heme proteins and models under ambient conditions have been studied by time-resolved spectroscopic techniques. Representative results are summarized in Table I (22-28). Equilibrium constants determined for the formation of nitrosyl complexes of met-myoglobin (metMb), ferri-cytochrome-c (Cyt111) and catalase (Cat) are in reasonable agreement when measured both by flash photolysis techniques (K= konlkQff) and by spectroscopic titration in aqueous media (22). Table I summarizes the several orders of magnitude range of kon and kQs values obtained for ferri- and ferro-heme proteins. Many k0f[ values were too small to determine by flash photolysis methods and were determined by other means. The small values of kQ result in very large equilibrium constants K for the... 

The first electrochemical studies of Mb were reported for the horse heart protein in 1942 (94) and subsequently for sperm whale Mb (e.g., 95) through use of potentiometric titrations employing a mediator to achieve efficient equilibriation of the protein with the electrode (96). More recently, spectroelectrochemical measurements have also been employed (97, 98). The alternative methods of direct electrochemistry (99-102) that are used widely for other heme proteins (e.g., cytochrome c, cytochrome bs) have not been as readily applied to the study of myoglobin because coupling the oxidation-reduction eqiulibrium of this protein to a modified working electrode surface has been more difficult to achieve. As a result, most published electrochemical studies of wild-type and variant myoglobins have involved measurements at eqiulibrium rather than dynamic techniques. 

The preceding equations have not yet been used as basis for the analysis of any actual titration curve of a protein. It should be feasible soon to apply the theory to the titration curve of myoglobin, the complete three-dimensional structure of which should be available soon (Kendrew et al., 1961). This will permit exact localization of all titratable groups on the protein molecule and, hence, the theoretical evaluation of the from assumed intrinsic pK s. It is not to be expected that these values will accurately reproduce the titration curve, but it is to be expected that the cause of observed deviations will be relatively easy to determine. Essentially three possibilities would be looked for in such a first application of the theory. 

It should be noted that four of the anomalous imidazole groups are the four groups by which the heme iron atoms are attached to the protein. These cannot be titrated as long as the hemes remain attached. The other groups must be simply buried in the interior. Such groups occur in myoglobin (see below), as well as in hemoglobin. 

Special interest attaches to the titration of sperm whale myoglobin because the three-dimensional structure of this protein is well on the way to being completely elucidated (Kendrew ei al., 1961). The speculative structural features, which have been invoked to explain titration data that do not conform to expectation, will in this protein soon be subject to actual test. 

A titration curve for sperm whale myoglobin has been reported by Bres-low and Gurd (1962). The most striking feature is that it exhibits a time-dependent acid denaturation, which resembles that observed for the similar protein hemoglobin. To elucidate the physical nature of this reaction, emphasis was placed on the back titration to neutral pH of denatured protein. As in the case of hemoglobin (mentioned earlier), there are two major differences between the titration curves of native and denatured myoglobin, as shown by the data of Table XV. 

FIG. 13. (a) Aromatic carbon region in the convolution-difference natural abundance C spectra of myoglobins recorded at 15-18 MHz, under conditions of noise-modulated off-resonance decoupling and 32768 accumulations per spectrum. See ref. 683 for line assignments, (b) The eight titratable carbons of the histidine residues (plus C of Tyr-103, peak 27) of horse cyanoferrimyoglobin at 38°C. 

The enthalpy of ionization of the titratable histidine groups in myoglobin is 7.5 kcal mol-1 (Antonini and Brunori, 1971). These groups are involved at the isoelectric point level. The solvent effect on the pKa of histidine has been measured. From a value of 6.1 in pure water, it decreases to 5.85 in the mixed solvent, at +20°C. The conditions (AH buffer > AH of the pi) for isoelectric focusing are therefore satisfied. 

The titration curve of myoglobin with oxygen is a hyperbola, as shown in Eigure 6-2 of the form ... 

Several ways to consider flexibility exist. A simple and fairly straightforward approach is to use several crystal structures of the given protein if these are available. For example, Bashford and Karplus considered two structures of lysozyme, and Bashford et al. considered five structures of myoglobin. These authors pointed out that the overall titration results are relatively insensitive to differences in those structures. 

No such complete body of data on the oxygen Bohr effect is available for any other protein as for horse hemoglobin, but we shall present such facts as there are. A differential titration of myoglobin is lacking, but... 

The importance of accurately determining pKa values of titratable sites of proteins is supported by the large number of studies pertaining to their calculation. In addition to method development " " " and review articles,some of the systems most recently studied are noted here a-chymotrypsin, bacteriorhodopsin, myoglobin, ribonuclease ribonuclease protein BPTI, lyso-... 

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