Selective Precipitation Using Metallic And Polyphenolic Heteropolyanions
Under strongly acid conditions, inorganic and some organic strong anions—known as heteropolyanions—ably precipitate proteins. Inorganic anions used under acid conditions for this purpose include perchlorate, tungstate, phosphotungstate, molybdate, phospho-molybdate, tungstosilicate, and ferrocyanide. Inorganic ions (used under acid to neutral conditions) include sulfosalicylate, picrate, and diverse plant polyphenols and tannates (also see Critical Parameters).
Prior to overt precipitation, protein molecules under strongly acidic conditions are acid-expanded and remain soluble. When the heteropolyanions are introduced and allowed to bind, the protein molecules are forced back to a compact, poorly hydrated conformation. Molecular expansion and contraction of the protein in solution, which leads to precipitate formation, may be followed by biophysical tools (Fink, 1995). Driven further with additional heteropolyanions, such as perchlorate and tungstate in ~0.2% to 2% or 3% concentration, proteins coprecipitate with these anions in dense aggregates that easily settle or centrifuge down.
In some procedures for precipitating proteins, perchloric acid (HClO4) is the precipitating agent of choice because it is ultraviolet-transparent above 250 nm. Perchloric acid precipitates whole protein molecules, but not their low-molecular-weight fragments— amino acids and oligopeptides. This is the basis for analysis with many proteolytic enzymes—at the end of the proteolytic cleavage assay, whole or intact proteins are precipitated out by a few percent HClO4. Proteolytic fragments are left in the supernatant for subsequent A280 measurement, giving a rather direct measure of the amount of hydrolysis that occurred before adding the HClO4.
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