title: Myoglobin extraction from mammalian skeletal muscle and oxygen affinity determination under physiological conditions.
abstract: An accurate determination of myoglobin (Mb) oxygen affinity (P50) can be difficult due to hemoglobin (Hb) contamination and autoxidation of Mb to metMb which is incapable of binding oxygen. To reduce Mb autoxidation, P50 is often measured at refrigerated temperatures. However, the temperature dependent shift in Mb oxygen affinity results in a greater oxygen affinity (lower P50) at colder temperatures than occurs at physiological temperature (ca. 37-39°C) for birds and mammals. Utilizing the temperature dependent pH shift of Tris buffer, we developed novel methods to extract Mb from vertebrate muscle samples and remove Hb contamination while minimizing globin autoxidation. Cow (Bos taurus) muscle tissue (n=5) was homogenized in buffer to form a Mb solution, and Hb contamination was removed using anion exchange chromatography. A TCS Hemox Blood Analyzer was then used to quickly generate an oxygen dissociation curve for the extracted Mb. The oxygen affinity of extracted bovine Mb was compared to commercially available horse heart Mb. The oxygen affinity of extracted cow Mb (P50=3.72±0.16 mmHg) was not statistically different from commercially prepared horse heart Mb (P50=3.71±0.10 mmHg). With high yield Mb extraction and fast generation of an oxygen dissociation curve, it was possible to consistently determine Mb P50 under physiologically relevant conditions for endothermic vertebrates.
authors.affiliation: Department of Marine Biology, Texas A&M University at Galveston, Galveston, TX 77551, USA; Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA. Electronic address: traywright@gmail.com.
authors.forename: Traver J
authors.initials: TJ
authors.lastname: Wright
authors.affiliation: Department of Marine Biology, Texas A&M University at Galveston, Galveston, TX 77551, USA; Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA. Electronic address: davisr@tamug.edu.
authors.forename: Randall W
authors.initials: RW
authors.lastname: Davis
mesh_terms: D000595:Amino Acid Sequence; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D002417:Cattle; D006736:Horses; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D009211:Myoglobin; D010100:Oxygen
keywords: Anion exchange chromatography; Myoglobin; Oxygen affinity; Oxygen dissociation