酶的分離工程ppt課件(編輯修改稿)
2025-02-01 20:18 本頁面
【文章內(nèi)容簡介】 e! Salting out (Can be used for fractionation) Beyond a certain ionic strength（ ） , the charged molecules are quickly precipitated because the excess ions (not bound to the protein) pete with proteins for the solvent. Saltingout effect: ions take ?all‘ water, expose the nonpolar surface。 solubility decrease! ++++++++ ++++++++ O H H + OHH+O H H+Precipitation ?Protein molecules are dehydrated by strong salt solution. ?The charges of protein molecules are neutralized. At low concentrations, the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting protein into the solution and enhancing the solubility of protein. This is monly known as saltingin. However, as the salt concentration is increased, a point of maximum protein solubility is usually reached. Further increase in the salt concentration implies that there is less and less water available to solubilize protein. Finally, protein starts to precipitate when there are not sufficient water molecules to interact with protein molecules. This phenomenon of protein precipitation in the presence of excess salt is known as saltingout. Used to selectively precipitate proteins, often with (NH4)2SO4 which is cheap, effective, does not disturb structure and is very soluble. Salting out (Ammonium sulfate precipitation) Salt concentration is indicted in Percentage saturation（飽和度） Volume of saturated (NH4)2SO4 Saturation ratio＝ Total solution volume How to achieve desired percentage of ammonium sulfate？ Add…… saturated solution dry powder How to making X% solution from Xo% solution? The effect of salt on different proteins may differ: Certain proteins precipitate from solution under conditions in which others remain quite soluble. Once the protein is precipitated (not denatured) – can separate by centrifugation ? pellet can be redissolved in buffer for further purification ? Which protein will ppt first? (hydrophobic or hydrophilic?) Fractional salting out——Different proteins precipitate at different salt concentration. serum globulin albumin (NH4)2SO4 50% saturation saturated precipitate precipitate Salting out curve ?protein(mg) or enzyme activity 10 20 30 40 50 60 70 80 90 100(NH4)2SO4 percentage of saturated In brief, the procedure goes as follows: 1)obtain protein solution of interest 2) add (NH4)2SO4 to a chilled, stirring solution 3) allow to stir for 1530 minutes 4) collect precipitated protein by centrifugation 5) redissolved in buffer for further purification ?Important factors: ?1) ionic strength ? S = solubility of the protein Ks： saltspecific constant β : idealized solubility I : the ionic strength of the solution log S = β Ks I ?2) pH: ≈ pI ?3) temperature ?low ionic strength ,T. protein solubility ?high ionic strength ,T. protein solubility ?4) protein concentration: moderate 2. Precipitation with anic solvents ——Decrease in dielectric constant ? Organic solvent decreases the water activity and the dielectric constant of the solution, which then decreases the solubility of the protein and
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