bio lab 5 - Biology 05LA Wiinter Quarter 2008 Lab 5 page 1...

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Biology 05LA – Wiinter Quarter 2008 Lab 5 – page 1 LAB #5: ENZYMES Enzymes are organic catalysts that participate in the chemical conversion of one organic molecule to another. The molecule that is acted upon by an enzyme is known as its substrate and the molecule that is formed by the reaction is referred to as its product. Enzymes regulate the rate of a reaction without being chemically changed in the course of the reaction. Thus, one molecule of an enzyme could theoretically catalyze an infinite number conversions of substrate to product. When enzymes were first discovered, they were given a wide variety of names. For example, the enzyme that initiates the digestion of starch in the mouth, salivary amylase, was formally known as ptyalin. The enzyme from the intestinal mucosa that splits lactose to glucose and galactose, beta- galactosidase was formally referred to as lactase. Currently, enzymes are named systematically by what they do. The ending "ase" identifies a substance as an enzyme. This ending is preceded by a stem which indicates a specific substrate or the general nature of the substrate or the type of action the enzyme catalyzes. For example, succinic dehydrogenase removes two atoms of hydrogen from succinic acid, while hydrolases insert a molecule of water across a bond and thereby hydrolyze a compound. Most enzymes are proteins and are sensitive to environmental conditions. Enzymes, like most proteins, can be irreversibly damaged or denatured by very high temperatures or pH extremes. There are fascinating exceptions to these general rules: enzymes whose critical component is not protein, enzymes from organisms that thrive in super-heated ocean volcanic vents, and enzymes that operate quite well at very acidic or very alkaline pH conditions normally viewed as deadly for all proteins and all life. All enzymes, even the unusual ones, have conditions of temperature, pH, concentration of enzyme and concentration of substrates that promote their most efficient activity. In today's exercises, we will examine bovine intestinal alkaline phosphatase. This enzyme normally operates in the intestines of cattle, away from the acidic stomachs (cattle have more than one), breaking down a variety of phosphorylated compounds found in their food. Alkaline phosphatase can hydrolyze the artificial substrate p-nitrophenylphosphate, giving us a convenient way to make quantitative measurements of its activity under various conditions. The basis for this convenience is derived from the fact that p-nitrophenylphosphate (pNPP) is a chromogenic substrate for alkaline phosphatase (and most other phosphatases). This means that an aqueous solution of pNPP, which is colorless, turns yellow when de-phosphorylated by alkaline phosphatase to form p-nitrophenol. This reaction is summarized as follows: a l k a l i n e p-nitrophenylphosphate (colorless) + H 2 O p-nitrophenol (yellow) + Pi (inorganic phosphate) phosphatase Further, a quantitative measure of the amount of yellow product (p-nitrophenol) generated in a
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This note was uploaded on 04/07/2008 for the course BIO 5A taught by Professor Zhu/cardullo/rao during the Winter '08 term at UC Riverside.

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bio lab 5 - Biology 05LA Wiinter Quarter 2008 Lab 5 page 1...

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