BIOS 2011 Lab 1 Enzymes

Methylene blue is normally blue in its oxidized form

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Unformatted text preview: ase’s active site for a time, preventing the enzyme from binding to succinic acid. The binding is not permanent, but malonic acid can bind and unbind to the enzyme repeatedly, disrupting the enzyme’s normal activity and lowering the rates of both fumarate and FADH2 production. Methylene Blue (MB) is a dye that has a number of uses in biology and medicine. In the experiment today, you will use it as an indicator of Succinic Dehydrogenase activity. Methylene blue is normally blue in its oxidized form, but becomes clear when it is reduced by the addition of two protons and two electrons. This reduced form is called Methylene White (MBH2). Methylene blue can be reduced in place of FAD, by Succinic Dehydrogenase in the presence of succinic acid substrate, and the test tube will lose its blue color. if the reaction becomes inhibited, less color change occurs. Note that the clear Methylene white can readily lose its electrons to oxygen from 4 Exercise 1 Enzymes the atmosphere and become re ­oxidized to blue. This re ­oxidation can be stopped by applying a surface coat of mineral oil on top of aqueous solution in your test tube. α Amylase and the Starch Iodide Test: α Amylase is named for its ability to break down Amylose the linear chains of glucose polymer that is part of starch molecules. It catalyzes the hydrolysis of every other a ­1,4 glycosidic bonds between the glucose monomers in amylose, with the end product being mostly maltose, a 12 carbon sugar that is a dimer of glucose, and some maltotriose, an 18 carbon trimer of glucose. Maltose then can be broken down to glucose by other enzymes, called Maltases. The 3D structure of eukaryotic α ­Amylase includes one calcium ion (Ca++) which helps each a ­Amylase polypeptide to fold properly and one allosteric site one for a chloride ion (Cl ­) in which chloride activates the enzyme. Both these ions are needed for the enzyme’s activity, so a ­Amylase works best in saline solutions that have a small amount of calcium. In mammals, a ­Amylase a enzymatic protein secreted by the cells of salivary glands and pancreas, along with chloride ­rich saline containing some calcium and buffered with phosphate. Our α ­Amylase comes in commercial powdered form isolated from pig (Sus scrofa domesticus) pancreas. Our assay for a ­Amylase activity is based testing for the presence of the starch substrate, to determine when it is all gone. We will be using the Starch Iodine Test. The indicator solution you will use is a yellow solution of elemental iodine in potassium iodide and potassium chloride (called IKI). When amylose starch combines with iodine, t...
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This note was uploaded on 02/09/2012 for the course BIOLOGY 102 taught by Professor Anderson during the Spring '11 term at Harvard.

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