What Are Enzyme Kinetic Assays?

What Exactly Are Enzyme Assays And Why Do We Use Them?

An enzyme assay is the name given to any laboratory technique that measures enzyme activity within a sample.

Enzyme assays can be used for a variety of purposes, which include identifying the presence of an enzyme, investigation of specific enzyme kinetics or the activity of inhibition within a sample.

Measuring Enzyme Activity

When it comes to measuring enzyme activity, both qualitative and quantitative methodologies can be used.

Qualitative assays are used to identify the presence (or absence) of a particular enzyme. On the other hand, a quantitative assay can be performed to determine the amount of the target enzyme that is present within a sample.

For qualitative assays, the use of coloured compounds is typically enough to confirm or deny the presence of the target enzyme. However, to ensure reproducibility and avoid operator error, using an appropriate instrument such as a colorimeter or photometer may be more appropriate than visual confirmation alone.

Alternatively, quantitative assays are used in cases where instruments operating in the visual range are not suitable or determination of enzyme concentration of kinetic mechanisms are of interest.

Types of Enzyme Assay

There are two types of enzyme assay, which can be split into two; continuous and discontinuous assays.

1. Continuous Enzyme Assay

In continuous assays, the course of the reaction is continually followed until completion.

Sometimes referred to as ‘endpoint assays’, enzyme activity is measured via the quantity of substrate consumed, or the amount of product formed during the reaction over a fixed period of time. Both values are directly proportional to the concentration of enzymes present in the sample.

Examples of continuous assays include spectrophotometry, calorimetry, chemiluminescence and fluorimetry. In these methods, the progress of reactions are measured by light or heat.

2. Discontinuous Enzyme Assay

In contrast to continuous enzyme assays, discontinuous assays are performed when samples are taken at set intervals. This form of enzyme assay directly or indirectly measures changes in substrate or products over time, to understand how the reaction rate changes.

Examples of instrumentation used during discontinuous enzyme assays include radiometric assays as well as chromatographic assays such as HPLC or TLC.

Comparing the two methods, the continuous enzyme assay method is typically the easiest to perform and can give whilst discontinuous enzyme assays are used in cases where higher precision or complex sample matrices are present.

Factors That Affect Enzyme Assay

In order for an enzyme assay to remain accurate, controlling external factors so they do not influence the outcome of the assay is crucial.

1. pH

All enzymes have an optimum pH range where their rate of reaction is highest. Anything too far out of the optimum range will cause denaturation and a reduction in reaction rate.

2. Temperature

Generally speaking, as temperature increases, so does the reaction rate of an enzyme. However, once temperature exceeds a certain threshold, reaction rates can drastically drop. This is due to the effect that temperature has on the bonding of the reaction site.

3. Substrate Saturation

Increase in substrate concentration will increase the rate of reaction, but only up to a certain point. At the point of saturation, the reaction rate is at its upper limit and will not increase, regardless of how much substrate is added.

4. Salt Concentration

Enzymes typically work best in low salt concentration environments. Increase in salt concentration causes interference with bonds, which impact the structure of the bonding site and the resulting reaction rate.

Enzyme Assay Use Cases

Enzyme assays cover a wide range of real life use cases. A couple of examples of enzyme assays include the following:

1. Alcohol Concentration

The measurement of short chain alcohols (methanol, ethanol, propanol) can be useful to determine a range of properties, such as the level of alcohol in food and drink, or metabolism rates.

Alcohol can be metabolized via many pathways, and is broken down by two enzymes – alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) – so measuring the properties of these enzymes can give a wealth of information.

2. Lactate Enzymatic Assay

Lactate is a metabolite produced by the breakdown of glucose in humans and animals via anaerobic metabolism. It forms when NADH is oxidized to NAD+, due to the reduction of pyruvate to lactate, which is catalyzed by the enzyme lactate dehydrogenase (LDH).

Measuring the activity of LDH can help understand information about the process of the metabolism process.

Performing Enzyme Assays With Photopette® Cell

In traditional enzyme assays, performing measurements via a spectrophotometer typically means obtaining samples in a cuvette and repeating.

With Photopette® Cell, our handheld device has been designed for a sampling free workflow to increase efficiency and decrease cross-contamination. Dedicated to measurements at 340 nm, 570 nm and 600 nm, Photopette® Cell is the ideal instrument for enzyme assays.

FAQs

• Why do we need to measure enzyme activity?
  Performing enzyme assays provide a wealth of information related to enzyme activity. This includes the identification of an enzyme, the concentration of enzyme and the rate of reaction within a sample.
• What is an endpoint enzyme assay?
  An endpoint assay is an assay method that is run for a predetermined amount of time. The amount of substrate consumed and/or the quantity of product formed is measured over the course of the reaction.
• What is a kinetic enzyme assay?
  In the kinetic assay method, the progress of the reaction is continuously measured as substrates are converted into products. Changes in concentration of both substrate and product cause shifts in measurements.
• What is the difference between endpoint and kinetic?
  The main difference between the two methods is how measurements are taken. In the endpoint assay, a final measurement is taken to measure the total amount of substrate/products. In the kinetic assay method, multiple measurements are taken over the course of the reaction.
• How do you measure endpoint activity?
  Endpoint measurements can be performed qualitatively, via visual assessment, or quantitatively via instruments such as spectrophotometer or calorimeters. Photopette® Cell by Tip Biosystems is a handheld device which can be used to measure an endpoint.