Spectrophotometric Determination of Cytochrome C

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TOPIC SPECTROPHOTOMETRIC DETERMINATION OF CYTOCHROME C ABSTRACT We devised and carried out an experiment to determine the cytochrome c concentration of a sample solution. A graph of variation in absorbance with wavelength of another solution of fixed concentration was used to determine the wavelength of maximum absorbance (□max). Another graph of the variation in absorbance at □max with concentration was used to quantify for the cytochrome c in the solution ‘X’. The slope of the second graph was then determined. A value of A(1% 1cm) was obtained. INTRODUCTION Cytochromes are proteins with an iron-containing group (heme) capable of existing alternatively in a reduced form (Fe2+) and an oxidised form (Fe3+) form and are specifically involved in electron transport in mitochondria. (Tortora & Grabwoski, 2000). Several cytochromes are involved in the electron transport chain and includes cytochrome a (cyt a), cytochrome a3 (cyt a3) cytochrome b (cyt b), cytochrome c1 (cyt c1) and cytochrome c,(cyt c). The electron carrier cytochrome c resides in the intermembrane space between the outer and inner mitochondrial membranes and helps the electron transport chain to convert energy as NADH to energy as the hydrogen ion electrochemical gradient across the mitochondrial inner membrane. Although cytochrome c is soluble protein of relative molecular mass 12,270, it cannot escape from the intermembrane space into the cytosol because porin, the channel of the outer mitochondrial membrane, only allows solutes of molecular mass ≤ 10,000 to pass. (Bolsover et.al, 2004). Although cytochrome c is essential for mitochondrial function, it has another deadly role. If cytochrome c comes into contact with a class of cytosolic enzymes called caspases, it activates them, turning on the process of cell suicide called apoptosis. Under certain conditions, porin can associate with other

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