Isoelectric pointThe precipitation method is to useproteinAt isoelectric pointsolubilityIt is a method for separating proteins with the lowest and different isoelectric points.
stayIsoelectric pointWhen the protein moleculeZwitterionForm exists, its moleculeNet chargeIs zero (that is, the positive and negative charges are equal). At this time, the protein molecular particles in the solution do not repel each other with the same charge, and the moleculesForceWhen the protein is at the isoelectric pointsolubilitySmallest, easiest to formprecipitate。Many of the isoelectric pointsphysical propertyasviscosity、Expansibility、osmotic pressureThe waiting time becomes smaller, which is beneficial toSuspensionFiltering of.
matters needing attention
Announce
edit
1. Different proteins have differentIsoelectric point。stayProduction processAccording to the separation requirements, the foreign proteins other than the target products should be removed;If the target product is also a protein, and the isoelectric point is high, the foreign protein lower than the isoelectric point can be removed first, such asCytochrome C[1]Its isoelectric point is 10.7. During the extraction and purification of cytochrome C, adjust pH=6.0 to remove acidic protein, adjust pH=7.5~8.0 to remove alkaline protein.
2. The same protein has different isoelectric points under different conditions.staySalt solutionMedium, if the protein combines morecation, the pH value of isoelectric point increases;Because after combining cation,positive chargeRelative increase, only when the pH value increases can the isoelectric point state be reached, such asinsulinThe isoelectric point in aqueous solution is 5.3, and the isoelectric pointZinc saltOf water -acetoneThe isoelectric point in the solution is 6;If the concentration of zinc salt is changed, the isoelectric point will also change.If more protein is boundanion(such as C1 -, SO42 -, etc.), the isoelectric point moves to a lower pH value becausenegative chargeIt is relatively increased, and only by reducing the pH value can the isoelectric point state be reached.
3. Requirements for pH value of target drug ingredients.Try to avoid direct use in productionstrong acidOr strong base to adjust pH value, so as to avoid local hyperacidity or alkalescence, which may cause the protein or enzyme of the target drug componentdenaturation。In addition, the acid or base used to adjust the pH value should be compatible with the salt in the original solution or the salt to be added, such assulphuric acidFor ammonium, sulfuric acid orammoniaAdjust the pH value, if the original solution containssodium chlorideAvailablehydrochloric acidorsodium hydroxideAdjust the pH value.In short, try not to increaseNew substancesIs the principle.
4. Because various proteins still have certainsolubility, so that the precipitation is incomplete, and mostIsoelectric point of proteinThey are very close to each other, so when the equal point electricity is used alonePrecipitation methodWhen the effect is not ideal, a combination of several methods can be considered to achieve precipitation separation.
