Isolation and characterization of the enzyme alkaline phosphate from escherichia coli: protein purification. 2) why is it...

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Chemistry

Isolation and characterization of the enzyme alkaline phosphatefrom escherichia coli: protein purification.
2) why is it necessary to dialyze the enzyme after ammoniumsulfate precipitation?

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Ammonium sulphate fractionationProteins are soluble in aqueous media because they havehydrophilic amino acid sidechains facing outwards that caninteract with water These are provided by the basic amino acidsarginine histidine arginine and lysine the acidic amino acidsaspartate and glutamate and the neutral hydrophilic amino acidsasparagine glutamine serine threonine tyrosine andcysteineAny compound that interferes with these interactions betweenamino acid sidechains and water by reducing the available waterwill reduce the solubility of the protein As interactions withwater become less marked so proteinprotein interactions becomemore important and the protein will aggregate and come out ofsolution Provided that the temperature is maintained low enougharound 4C the protein is not irreversibly denatured but theprecipitate can be redissolved in bufferA number of different methods can be used to reduce hydrophilicinteractions and precipitate out proteins reasonably selectivelyincludingSalting out with ammonium sulphateSelective precipitation with an organic solvent that bothreduces available water and also decreases the dielectric constantof the solution Ethanol fractionation is commonly used to separateprotein fractions from blood plasma Historically acetoneprecipitation was an important way of obtaining a dry powder thatcould be stored for some time without losing activitySelective precipitation using nonionic polymers such aspolyethylene glycol which both reduce the available water and mayalso interact directly with some of the proteins in the mixture inmuch the same way as do the polymers used in gel filtrationAmmonium sulphate is highly hydrated and a concentratedammonium sulphate solution reduces the available water veryconsiderablyThe diagram on the right shows two proteins with theirhydrophilic regions coloured blueThe protein on the left hasrelatively few hydrophilic regions and hence will aggregate andprecipitate at See Answer

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