1. Creatine kinase (CK) catalyzes the conversion of creatine and utilizes adenos
ID: 133781 • Letter: 1
Question
1. Creatine kinase (CK) catalyzes the conversion of creatine and utilizes adenosine triphosphate (ATP) to create phosphocreatine (PCr) and adenosine diphosphate (ADP). You are interested in extracting creatine kinase from the livers of goats in various age groups for your study. After obtaining the lysed tissues, you are planning the strategy for the initial purification a) You decide to implement centrifugation as the first step of your extraction. At what speed, as in RCF, will CK precipitate? How can temperature play a role in your product yield? (S pts) b) After centrifugation, you plan to use ammonium sulfate precipitation to isolate CK from your clarified homogenate. Briefly, in 2-3 sentences, explain how ammonium sulfate salt precipitates your target protein, why increasing salt content (% saturation) could lead to an over-precipitation and compromise this purification step? (5 pts)Explanation / Answer
a) In a study for brain isoenzyme of creatine kinase by John.B.Armstrong et al., the homogenate was centrifuged for 40min. at 2800g / RCF (5000rpm) at -100C after adding NH4Cl, adjusting pH to 8.5 with NH4OH at 20C. The centrifugation was performed after adding ethanol which was at - 200C at 300ml/h until the concentration is 55%, and it was again was stirred at -100C for 30min before centrifugation.
In this study, the temperature was kept always at -100C and at no time did it rise above 20C. The effect of temperature on handling enzyme is that the enzyme is kept in low temperatures as the activity could lose at higher temperatures.
b) Ammonium sulfate precipitation is one of the most commonly used methods for large scale protein purification and fractionation that can be used to separate proteins by altering their solubility in the presence of a high salt concentration.
The solubility of proteins varies according to the ionic strength of the solution, thus according to the salt concentration. At low ion concentrations (<0.5 M), the solubility of proteins increases with increasing salt concentration, an effect termed salting in. As the salt concentration is further increased, the solubility of the protein begins to decrease. At a sufficiently high ionic strength, the protein will precipitate out of the solution, an effect termed salting out. When the ammonium (NH4+) and sulfate (SO42?) ions are within the aqueous solution they are attracted to the opposite charges evident on the compound that is being purified. This attraction of opposite charges prevents the water molecules from interacting with the compound being purified, leading to the precipitation or salting out.
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