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Analysis of Extracellular hemoglobin by UV-Vis Spectroscopy (CAT#: STEM-MB-0883-WXH)
Introduction
Extracellular hemoglobin, resulting from hemolysis or exogenous infusion, exerts toxic effects that are major components in the pathogenesis of many diseases and iatrogenic situations, including hemolytic anemias and transfusion-induced intravascular hemolysis, preeclampsia, intraventricular hemorrhage, sickle cell disease and chronic inflammatory leg ulcers, and infusion of recombinant hemoglobin. The pathogenesis involves one-electron reactions between oxy-Hb, its downstream metabolites heme, iron, ROS and free radicals (NO, superoxide etc.) and exposed tissue components. Besides, release of extracellular hemoglobin can result in the removal of beneficial free radical species (e.g. nitric oxide) and/or the production of reactive free radicals on the globin protein itself.
Principle
UV-Vis spectroscopy is an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. This property is influenced by the sample composition, potentially providing information on what is in the sample and at what concentration. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.
Applications
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of diverse analytes or sample, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.
Procedure
1. Calibrate the Spectrometer
2. Perform an Absorbance Spectrum
3. Kinetics Experiments with UV-Vis Spectroscopy
2. Perform an Absorbance Spectrum
3. Kinetics Experiments with UV-Vis Spectroscopy
Materials
UV/VIS Spectrophotometer
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