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Author Topic: ENZYME KINETICS AND MONITORING ENZYME ACTIVITIES / REACTIONS.  (Read 5240 times)
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Francis Umeoguaju
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« on: April 10, 2010, 08:44:10 am »



Enzyme kinetic is a way of monitoring the activity, velocity, km, Vmax and other enzymatic parameters of an enzyme or a particular component of a protein extract. This may serve as a useful way for assaying for the protein of enzyme of interest especially when the protein exhibits peculiar kinetics under some specific condition such as temperature, pH or the presence or absence of a particular co Factor, substrate or inhibitor.

Of particular importance is the Vmax and Km. This can be a  useful parameter in comparing the activity of a particular extract or protein under different conditions of assay. Vmax may also be used identify a particular protein since proteins have characteristics Vmax  under specified condition.

The Kinetic parameters may also give insight to the physiological role of the enzyme especially in situations when it does not obey  michaelis-menten  equations such as the case of regulatory enzymes.


Monitoring the activity/kinetics of a protein or extract


Activity of an extract or enzymes can be done to determine the optimum condition for the action of the protein.

Usually the protein is subjected to different conditions such as varied pH, temperature, presence or absence of different cofactors. The intention usually, is to determine the condition that optimizes the activity of the protein of interest.

The activity of an enzyme constituent of an extract is determined by reacting known and constant concentration of the enzyme or extract and substrate under varying condition. The Kinetic parameter such as Vmax and Km of a protein is determined by incubating a constant quantity of the enzyme/extract with varying concentration  of the substrate under specific condition especially at those conditions where the enzymes has maximum activity.

Generally the reaction mixture is terminated by the addition of the same volume of 10% TCA or 4% perchloric acid at different time interval starting from time zero. The addition of TCA to the reaction mixture will stop all enzymatic activity and will precipitate the proteins present in it. The time zero sample usually serves as the blank.

The product of the reaction can then be estimated when all the reaction is completed. For proteolytic action of an extract, the product of the reaction which are usually free amino acid, protein and peptides can be determined by taking the absorbance at 280nm, the difference in absorbance of the blank from those of the extracts can be used to quantitate the product formed.

The product of the reaction can also be determined by estimating the alpha-amino acid content and the tyrosine soluble protein constituent of the terminated reaction mixture.

In some case, a progress curve which is graph of change in concentration of product or reactant against time is plotted for the various condition considered. The slope of the progress curve for the linear region of the curve gives the initial velocity. This can then be used to plot an activity curve of the enzyme under the various varying conditions considered.

The initial velocity can also be used to plot the Michealis-Menten curve for the case of enzyme kinetics study. From the Line weaver-Burk plot, the Km. and Vmax can be determine.

Sample experiment. Determination of the Vmax and km of the Reaction of Trypsin with casein.

REAGENTS AND PREPARATIONS
   0.2% casein (prepared by dissolving 40mg of casein in 20ml of buffer)
   
   0.66mg/ml Trypsin (this was prepared by dissolving 1.32mg of Trypsin in 2ml of 0.1N HCL)
   
   0.1M pH 7.6 phosphate buffer
   
   4% perchloric acid
   
   Lowry protein estimation reagent
   
PEOCEDURE
   Varying volumes of casein were pipette into 4 paired and labeled tubes. The volumes were made up to 1.95 by adding

   0.1N PO4 buffer. 0.05ml of the Trypsin solution was added to each tube. 2ml of perchloric acid were then added to a member of each paired tube immediately after the addition of the enzyme  to terminate the reaction.
   
   This tubes serves as the blanks of each pairs. The other reactions were terminated after 15 minutes. The reaction mixtures were centrifuged and the soluble tyrosine content, Absorbance at 280nm and the perchloric acid soluble alpha-amino content were determined.
   
   The velocity of the reaction was determined by dividing the change in the protein concentration of each pair by the reaction time (15mins). The Vmax and Km were extrapolated from the Lineweaver burk plot drawn.
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