The covalent structure of dog myoglobin

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2 )-allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii)-when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail (1-4).The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner.Our present experience (4) clearly indicates that histidine functions as the predominant ligand in the IMAC of proteins based on metal ions belonging to the latter part of the first series of transition metal elements (and zinc). Therefore, topography of the protein surface with respect to the location of histidine residues becomes critical for better understanding and exploitation of an IMA event (1, 4-6).A facile probe into the topography of histidyl residues could be quite useful, especially if it could provide information about their surface disposition. This information is required for the rational exploitation of IMAC for protein isolation. In a simple case, one has only to seek information about the surface accessibility of a putative electron donor grouping (7). However, the evaluation of an IMA event in the case of proteins replete in histidine residues may require more comprehensive analysis (8).We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique (9,10). IMAC can be used to ascertain several facets of the status of a histidyl...

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“…This number is diminished by two residues in the case of dog myoglobin: His-113 and His-116 are missing (30). The (Fig.…”

Section: Discussionmentioning

confidence: 99%

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Hemdan

Zhao

Sulkowski

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

267

127

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“…In the case of trout Mb, as shown here, S-nitrosation at a Cys residue appears to be responsible for the observed change in O 2 affinity. However, such a regulation mechanism cannot be of general significance, as most mammalian and avian Mbs lack Cys residues (7,39,8), in contrast to many fish and reptilian species that have Mbs containing several Cys (3). One could speculate that a high Cys content of the Mb and the ability to form S-nitrosated Mb may be beneficial in ectothermic animals like fish and reptiles, which are often subject to short-term fluctuations in tissue O 2 availability, regardless of the ability to adapt to long-term ambient hypoxia.…”

Section: Perspectives and Significancementioning

confidence: 99%

Allosteric modulation by S-nitrosation in the low-O₂affinity myoglobin from rainbow trout

Helbo

Fago

2011

American Journal of Physiology-Regulatory, Integrative and Comp

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Myoglobin (Mb) serves in the facilitated diffusion and storage of O₂ in heart and skeletal muscle, where it also regulates O₂ consumption via nitric oxide (NO) scavenging or generation. S-nitrosation at reactive cysteines may generate S-nitroso Mb (Mb-SNO) and contribute further to NO homeostasis. In being a monomer, Mb is commonly believed to lack allosteric control of heme reactivity. Here, we test whether in rainbow trout, a fast swimmer living in well-aerated water, the Mb-O₂ affinity is regulated by ionic cofactors and S-nitrosation. O₂ equilibria showed the lowest O₂ affinity ever reported among vertebrate Mbs (P₅₀ = 4.92 ± 0.29 mmHg, 25°C), a small overall heat of oxygenation (ΔH = -12.03 kcal/mol O₂), and no effect of chloride, pH, or lactate. Although the reaction with 4,4'-dithiodipyridine (4-PDS) showed 1.3-1.9 accessible thiols per heme, the reaction of Mb with S-nitroso cysteine (Cys-NO) and S-nitrosoglutathione (GSNO) to generate Mb-SNO yielded ∼0.3-0.6 and ∼0.1 SNO/heme, respectively, suggesting S-nitrosation at only one cysteine (likely Cys¹⁰). At ∼60% S-nitrosation, trout Mb-SNO showed a higher O₂ affinity (P₅₀ = 2.23 ± 0.19 mmHg, 20°C) than unmodified Mb (3.36 ± 0.11 mmHg, 20°C). Total SNO levels measured by chemiluminescence in trout myocardial preparations decreased after hypoxia, but not significantly, indicating that transnitrosation reactions between thiols may occur in vivo. Our data reveal a novel, S-nitrosation-dependent allosteric mechanism in this low-affinity Mb that may contribute to targeted O₂-linked SNO release in the hypoxic fish heart and be of importance in preserving cardiac function during intense exercise.

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“…It is perhaps useful to consider why the binding ability of the site is not necessarily completel3 Table 5. Antigenic sites ofsperm-whale Mb and substitutions within these regions in the other myoglobins The amino acid substitutions were based on the sequences given in the references cited: sperm-whale Mb (Edmundson, 1965; Romero-Herrera & Lehmann, 1974), finback-whale Mb (DiMarchi et al, 1978), killer-whale Mb (Castillo et al, 1977), chimpanzee Mb (Romero-Herrera & Lehmann, 1972), horse Mb (Dautrevaux et al, 1969;Romero-Herrera & Lehmann, 1974), sheep Mb (Han et al, 1972;V6tsch & Anderer, 1972), bovine Mb (Han et al, 1970), echidna Mb (Castillo et al, 1978), rabbit Mb (Romero-Herrera et al, 1976b), dog Mb (Dumur et al, 1976), cape-fox Mb (Jones et al, 1977) and chicken Mb (Deconinck et al, 1975). Residues in parentheses are part of the antigenic site only with some antisera.…”

Section: Factors Influencing the Reaction Ofan Antigenic Sitementioning

confidence: 99%

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species

Twining¹,

Lehmann²,

Atassi³

1980

Biochemical Journal

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The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.

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The covalent structure of dog myoglobin

Cited by 20 publications

References 27 publications

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Allosteric modulation by S-nitrosation in the low-O₂affinity myoglobin from rainbow trout

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species

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The covalent structure of dog myoglobin

Cited by 20 publications

References 27 publications

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Allosteric modulation by S-nitrosation in the low-O2affinity myoglobin from rainbow trout

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species

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Allosteric modulation by S-nitrosation in the low-O₂affinity myoglobin from rainbow trout