Myoglobin In The Utilization Of Oxygen In Animals Biology Essay

Myoglobin In The Utilization Of Oxygen In Animals Biology merchantmanvasMyoglobin is an oxygen-binding spheric protein which is vital in facilitating the acquisition and utilization of oxygen in animals. Myoglobin was isolated and purified from set up water buffalo paint a picture. The Myoglobin was isolated by cation- turn chromatography, and submersion of Myoglobin is measu reddened by spectrophotometry. Total immersion of protein was inflexible by per body-build Bradford protein assay. Iron analysis was performed by atomic engrossment spectrophotometry (AAS). Molecular clog of Myoglobin was resolved by running SDS-PAGE. A total amount of revenue ug Myoglobin was purified by the cation-exchange chromatography with a relative purity of 25.9%. The concent limit of the purified Myoglobin was determined to be 0.072 ug/uL. The molecular weight of Myoglobin was determined to be 18204 Da And 0.423 mg of weight-lift were detected in the acid digested extractIntroductionMyoglo bin is a globular protein which contains a single polypeptide chain of about 153 amino acids and an iron-porphyrin complex, or the hematin group (3). And it has a molecular weight of 16700 Da and 153 amino acids (4). The non-covalently resound heme group, which resides in the hydrophobic interior of the native globin chain, is able to unfold under acidic condition and consequently weakens the interaction surrounded by the heme group and the globin (5).Myoglobin is an oxygen-transport protein which can be found in muscle tissues of all mammles. Myoglobin is critical in mammalian cell in that it is responsible for storage and distribution of oxygen, and possibly carrying energy (4). Diving animals such(prenominal) as seals and whales have excessive amount of Myoglobin that help them travel undersea by storing and transport oxygen (9). It plays a significant role in the physiological function of kernel and skeletal muscle (2). Elevated expenditure of oxygen during exercise nece ssitates the production of myoglobin in red muscle and heart cells, and the transportation of oxygen by myoglobin from the sarcolemma to the mitochondria in vertebrate heart and red muscle cells. (1)Spectrophotometry studies the interaction of electromagnetic radiation with iotas, atoms and ions (10). It can shed abstemious on the physical and chemic properties by measuring the emission or absorption of electromagnetic radiation (10). Besides, it is also used to identify biomolecules from their individualistic absorption spectrum. In the meantime, spectrophotomoetry can quantitatively measure the concentproportionn of molecules in dissolver According to the Beer-Lambert law, the fraction of incident light absorbed by a solution at a tending(p) wavelength is indicative of the concentration of the absorbing species (10) . Tryptophan and tyrosine can absorb ultraviolet light, which accounts for the characteristic beefed-up absorbance of light at wavelength of 280 nm by most pro teins (9). Atomic absorption spectroscopy (AAS)can be used to determine the identity and concentration of chemical elements in the gaseous state by measuring the light radiation absorbed by the elements (10).Ion-exchange chromatography is a technique for separating biomolecules capable of being compound in electrostatic interactions (10). Molecules can be divide based on their sign and magnitude of net agitate at a revealn pH and formation of electrostatic linkages surrounded by thee rosin and the protein of interest (9). Ion exchange separations take place in pillars packed with an iron-exchange resin (10). Resins with bound non- noggin detergent groups argon cation exchanges whereas those with bound cationic groups are anion exchangers (9). Selection of ion exchange resin depends on what is to be purified, the pH to be used in the towboat, and the readiness of the functional group (10).Electrophoresis is the separation of proteins based on the crusade of the charged pro teins under the influence of an electric field (9). The migration of the protein depends on its shape, size, charge and chemical composition (10). An electrophoretic method, polyacrylamide gel electrophoresis (SDS-PAGE) can be used to estimate the purity and determine the molecular weight of the protein (9). intercession of the protein by the ionic detergent sodium dodecyl sulfate (SDS) can give it a uniform net charge, and protein can then be separated based solely on its mass (10).In this experiment, Myoglobin is extracted and purified by cation-exchange chromatography from ground water buffalo using Beffer A (20 nM, pH 5.6, KH2PO4) and pilot burner B (20mM, Tris buffer, pH 7.5). Absorbance of the eluent fractions is mensurable at 280nm and 417nm. Total amount of protein and the concentration of Myoglobin are determined using Bradford assay from BSA protein. Molecular weight of Myoglobin was determined by performing SDS-PAGE. Iron content in the acid digested extract is measura ble by atomic absorption spectrophotometry (AAS). A total amount of revenue ug Myoglobin was purified by the cation-exchange chromatography with a relative purity of 25.9%. The molecular weight of Myoglobin was determined to be 18204 Da. 0.757 mmol of iron and 39.5 nmol of myoglobin were detected in the acid digested extract, with a ration of 201.ResultsA total of five take ins were collected from a solution of 10.02g thawed ground buffalo mixed with 20.0mL, 20mM, pH 5.6, KH2PO4 (Buffer A) 1.0 mL of archaic extract (sample C), 1.0 mL of filtered extract (sample D), 2.0 mL of Buffer A sample solution (sample A), 2.0 mL Buffer B sample solution (sample B) and 5.0 mL acid digested extract solution. Chromatogram for the purification of myoglobin carried out by cation-exchange chromatography shows a great(p) peak from fraction number 5 to fraction number 12. (Figure 1). The formula also indicates another peak at fraction number 23, with little peaks at fraction number 19 and fracti on number 25 (Figure 1). Fraction number 23 has the highest 417 nm/ 280 nm (3.766) and the highest absorbance 90.278) at 280 nm (Figure 1).DiscussionWhen myoglobin was separated by cation-exchange chromatograph, purity of the myoglobin in the eluent fractions collected at specific volumes was examined by spectroscopy. Absorbance of Myoglobin, specifically, was measured at 417 nm and other proteins was measured at 280 nm, due to the presence of Tyrosine and Tryptophan Most proteins absorbs at a wavelength of 280 nm (10). Phophyrin has an absorbance spectrum of 414nm to 418 nm, and intensity and wavelength of the absorption can be influenced by the peripheral device substituents on the porphryin and the protonation state of the nitrogen atoms (6). Sincemyoglobin consists of a iron-containing heme prosthetic group with an iron-contained porphyrin ring (9), it can absorb at a wavelength of 417.Myoglobin has a PI value of 7, so it will have a net demonstrable charge when pH is below it s PI and a net interdict charge when pH is above its PI. When myoglobin is positively charged when buffer A (pH=5.6) is used to wash the column, it binds to a column containing negatively charged beads in cation-exchange chromatography. The positively charged Myoglobin can then be eluted by washing the column with buffers having higher pH value than 7 (Buffer B, pH= 7.5). Raising the pH of the mobiles phase buffer renders the Myoglobin less protonated and thus negatively charged. As a result, the Myoglobin is not be able to form an ionic interaction with the negatively charged stationary phase and then elutes from the column (10).144 ug Myoglobin was recovered form the column, which accounts for 10.7% of the myoglobin that was loaded onto the column. Loss of Myoglobin could be attributed to scattering spreading of Myoglobin and other contaminant proteins within the mobile phase, as a result of the increase in time length (9). Recovery of myoglobin can be improved by using narrow c olumns, longer column (7).The molecular weight of Myoglobin (18204 Da) obtained from the SDS-PAGE was similar to the literature value, 16700 Da (4), which indicated that the purified protein in the Buffer B sample was Myoglobin. The clear band generated from Lane B migrated the corresponding distance as the band from the Myoglobin standard, which further confirms that the purified protein was Myoglobin (Figure 3).The stoichiometric relationship between iron and hemoglobin is 201 instead of 11 according to the fact each molecule of Myoglobin has only one molecule of ion. The excessive amount of Fe might be exhibit in oxygen-carrying protein such as hemoglobin, which contains four heme prosthetic groups. Electron carriers in the mitochondrial respiratory chain have Fe incorporated in their prosthetic group, and examples of them include cytochomr c, ubiquinone and cytochrome oxidase. Besides, another hemeprotein, ferritin might also be present in the protein sample. Hemeprotein funct ions to stores and release iron atoms in biologically available form for use in heme and nonheme proteins and biochemical reactions (8).In conclusion, a ratio of 120 for Myoglobin and iron was obtained from the ground water buffalo. 144 ug Myoglobin was purified from the filtered extract with a relative purity of 25.9%. According to SDS-PAGE, the molecular weight of Myoglobin was determined to be 18402 Da.