Specific Activity and Its Importance in Protein Isolation

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Specific activity is a term used to measure the rate of reaction of an enzyme with a substrate. Specific enzyme activity (specific activity for short) is a measure of the purity of an enzyme solution and is quoted as units/mg. The value becomes larger as an enzyme preparation becomes purer since the amount of protein (mg) is typically less, but the rate of reaction stays the same (or may increase due to reduced interference or removal of inhibitors).

Measuring Enzyme Kinetics

Enzymes are proteins that act as catalysts in reducing the amount of activation energy for a reaction to occur. Activation energy is the energy that is required to get reactions "started" since many reactions do not occur very quickly (or occur at all) if they are thermodynamically possible. 

Enzymes catalyze specific reactions in a particular site (known as the active site) where the reactants bind and react to generate the products. Substrates have active sites that are specific only to them so that no other substances bind or react in the same site. Like a lock and key, each enzyme will catalyze only one specific reaction, with some exceptions. 

Specific activity is used in protein isolation methods to indicate the percentage of purification. It is important to know this unit when purchasing enzymes to indicate exactly how much of the pure enzyme you are getting.

When measuring enzyme kinetics (the rate of reaction of an enzyme with a substrate—or surface), specific activity is defined as the amount of substrate the enzyme converts (reactions catalyzed), per mg protein in the enzyme preparation, per unit of time. 

An example of specific activity would be:

  • The specific activity of the isolated enzyme was measured at 150 umoles/min/mg protein before purification and 800 umoles/min/mg, after purification.

Specific activity is an important measure of enzyme purity. Different batches of a pure enzyme should have the same values and even diluting an enzyme solution many times will have identical specific activity values even though there will be different enzyme activity values. This is because in calculating specific activity, the numerator (units/ml) and denominator (mg/ml) are affected equally.

Specific activity is very different from activity, but the calculation of specific activity is still dependent on the activity value. This means that the stated specific activity value also will be dependent on the enzyme unit definition.

Batches of enzymes that have lower than expected specific activity value may contain enzyme molecules that have become altered or mixed with impurities.

Factors Affecting Enzyme Activity

It is possible that one enzyme may have different measured activity values when measured in different labs. These are real differences in measured activity, not apparent differences caused by the use of different unit definitions.

The way an assay is carried out (the method of testing to determine the purity of enzymes) will influence the reported activity values. For example, enzymes are generally more active between temperatures of 37 degrees Celsius and 20 degrees Celsius, so if an assay is carried out at 21 degrees Celsius, the values will differ from the assays conducted at a temperature of 32 degrees Celsius.

Thus, the definition of the enzyme unit would be better expressed as 1 unit (U) is the amount of enzyme that catalyzes the reaction of 1 nmol of substrate per minute under standard conditions.