Enzyme Analysis

WHAT IS ENZYME ANALYSIS?

Enzyme analysis or “enzymatic analysis”, in blood serum, is the measurement of the activity of the specific enzyme in a sample of blood serum, usually to identify a disease. It is also vital for the study of enzyme kinetics and enzyme inhibition.

UNIT OF MEASURE

The quantity (concentration) of an enzyme in a given reaction (enzyme activity) can be expressed in molar amounts, as the case with any other chemical, or in terms of activity in enzyme units.

The activity of enzymes (known as “enzyme activity”) can be rated according to (or based on) the quantity of the substrate it can convert within a specific period, which is usually dependent on specified conditions.

Enzyme activity = moles of substrates converted per unit time = rate × reaction volume. 

The SI unit of enzyme activity is known as Katal.

1 Katal= 1mol/s.

Because a Katal unit is alarmingly large, a more practical and commonly used value is:

Enzyme unit (U) = 1 micromole/min.

1 enzyme unit = 16.67 nanokatals (approximately).

Enzyme activity reflected in Katal generally refers to that of the potential natural target substrate on which the enzyme is assumed to act on. Also, enzyme activity can be given as that of certain standardized substrates, like gelatin, and measured in gelatin digesting units (GDU), or milk proteins, and measured in milk clotting units (MCU). 

These units GDU and MCU are based on the rate at which one gram of the enzyme will digest gelatin or milk protein, respectively.

1 GDU = 1.5 MCU (approximately).

It should be noted that an increased amount of substrate will cause a corresponding increase in the rate of enzyme reaction until a peak is reached where further increase in the amount of substrate will not alter the rate of reaction, but the rate of reaction will level out because the amount of active sites available has stayed constant; a saturation point.

The specific activity of an enzyme is the activity of an enzyme per milligram of total protein (expressed in micromole per minute per milligram). Specific activity is another common unit and gives a measurement of enzyme purity in the mixture. 

Specific activity is the micromoles of product formed by an enzyme in a given amount of time (minutes) under given condition per milligram of total proteins. It is equal to the rate of reaction multiplied by the volume of reaction divided by the mess of total protein. 

The SI unit for special activity is Katal/kg, but a more practiced unit is micromole/mg × min. Specific activity is usually constant for a pure enzyme.

Errors may arise from differences in cultivation batches and/or misfolded enzymes and similar issues. An active site titration process can be done to eliminate these errors. This is a measure of the amount of active enzyme. The specific activity should then be expressed as micromole/mg × min active enzyme. 

Having known the molecular weight of the enzyme, the turnover number, or micromole product per second per micromole of active enzyme, can be calculated from the specific activity. The turnover number gives the number of times each enzyme molecule carries out its catalytic cycle per second.

ENZYME ANALYSIS METHODS

Different methods of enzymatic analysis are used in measuring the concentrations of substrates and products that exist and many enzymes can be analyzed using different methods.

Enzyme-catalyzed reactions can be studied using the types of experiments.

• Initial Rate Experiments

When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction abstains steady-state kinetics in which enzyme-substrate intermediates remain approximately constant over time and the reaction rate changes relatively slow.

• Progress Curve Experiments

Here, the kinetics parameters are determined from expressions for the species concentration as a function of time. Progress curve experiments are less common now.

• Transient Kinetics Experiments

In this experiment, reaction behavior is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. The experiment is too difficult to perform as it needs specialist techniques. It is not popular.

• Relaxation Experiments

An equilibrium mixture of enzyme, substrate, and product is perturbed, for instance by temperature, pressure, pH jump, and only the return to equilibrium is monitored. It is insensitive to mechanistic details and thus not typically used.

FACTORS WHICH AFFECT ENZYME ACTIVITY

Some physical factors affect enzyme activity. These include:

• Temperature.

Enzymes strive well in specific temperatures. At low temperatures, the number of successful collisions between the enzyme and substrate is reduced due to reduced molecular movement. The reaction is slow. 

Our human body is at a controlled temperature of 37°C. At this temperature, our body enzymes work best. However, this is not true of our body enzymes in all organisms. Higher temperatures disrupt the shape of the active size, thus reducing its activity or preventing it from working. 

The enzyme becomes denatured (lost its original nature), Proteins are twisted chains of amino acid molecules in the chain, with high temperatures, these forces are broken, and the enzyme, including its active site, will change shape. With this development, the substrate no longer fits, the rate of reaction will be affected, or the reaction will stop.

• Environmental pH

Enzymes are also affected by ph. Changing the ph of its surrounding will also alter the shape of the active site of an enzyme. 

Many amino acids in an enzyme molecule carry a change (positive or negative), within the enzyme molecule. Positively and negatively charged amino acids will attract (this is what contributes to the folding of the enzyme molecule, its shape, and the shape of their active site) a change in the pH. 

It will also affect the charges on the amino acid molecules, amino acids that attracted each other may no longer be, secondly, the shape of the enzymes, along with its active site, will change, the extremity of pH also denature enzymes, the changes may or may not be permanent. 

Enzymes work both inside and outside cells, in the digestive system where cell pH is between 7.0 pH and 7.4 pH. Cellular enzymes will work best within this ph range, but different enzymes, which have different optimum ph. 

The separation of hydrochloric acid dictates the optimum ph in the stomach, while the optimum ph in the duodenum is produced by the secretion of sodium hydrogen carbonate. 

The optimum ph of other enzymes in the digestive system are:

• 1. Pancreatic protease (trypsin)     7.5pH to 8.0pH
  2. Stomach protease (pepsin)  1.5pH to 2.0pH
  3. Salivary amylase 6.8pH
• Substrate Concentration

Enzymes work best in excessive substrate medium. As the concentration of the substrate increases, the activity of the enzyme increases. This means that more substrate can be broken down by the enzyme at the instance of more substrate availability. 

However, this activity can be hindered when a shortage of enzyme sets in, this means the enzyme cant’ work anymore faster even though there is plenty of substrates available. So when the number of enzymes, the no more substrate can be broken down, At this junction, the enzyme concentration is the limiting factor slowing the reaction.

• Enzyme Concentration

As the concentration of the enzymes is increases, the enzyme activity also increases. This means that as more enzymes are added, more substrate will be broken down, and again, an increase in enzyme activity under conditions can be hindered. 

When the amount of available enzyme surpasses the amount of substrate then no more substrate can be broken down, the contact of the substrate is exhausted, completely consumed, the substrate concentration is the limiting factor slowing the reaction.

CONCLUSION

Enzyme analysis is based on the measurement of how fast a given amount of enzymes will interact with a substrate to yield a product. 

Examples are specialized proteins and biological catalysts responsible for activating chemical reactions within cells. They carry out chemical reactions amazingly, at a high rate within the mild conditions of temperature, pH, and pressure of cells. Their activity is with remarkable efficiency and specificity. 

They function using the active sites, a specific place where substrates are bound, in the enzyme-substrate complex. Enzyme activity is determined by measuring the number of products formed, or substrate consumed in a reaction in a given time. Substrates are the substances on which enzymes act.

FAQs

• What is an enzymatic analysis used for?

Enzyme analysis serves two different purposes, one of which is to identify a specific enzyme, to confirm its presence or absence in a distinct specimen, like an organism or tissue, as well as to determine the amount of the enzymes in the given sample.

• What are the three factors of enzymes?

Enzymes help in speeding up the chemical reactions in the human body. They bind to molecular (substrate) at active sites and alter them in specific ways. They are essential for digestion, respiration, muscle and nerve functioning, etc, in the human body system.

• What is the enzyme activity of an enzyme?

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume.

• What four things can affect the way enzymes work?

Several factors affect the rate at which enzyme reactions work. These include temperature, pH, enzyme concentration, and substrate concentration; as discussed earlier.

• What are the features of enzymes?

Enzymes are highly specific for a particular substrate. They also remain unchanged during every subsequent reaction. Furthermore, they are very effective, catalyzing about 1 in every 10,000 molecules of substrate per second. Finally, they do not affect the equilibrium constant; K.