Enzymatic analysis. Part 2

April 25 @ 12:00 – 13:00

Introduction to Lesson 5

We have previously studied the structure of proteins, and, among the different proteins, the antibodies. Now is the turn for another important proteins in analysis: the enzymes. They are very useful biomolecules for analysis in different field, ranging from food safety, environmental monitoring, but mostly in clinical analysis. 

You can take a look in any personal blood analysis in routine medical checking. You will fins a lot of parameters that are measured, including Creatinin,  Glucose,   Uric Acid, Urea, Colesterol,   Aspartate aminotransferase (GOT  or  AST),    Alanine aminotransferase (ALT or GPT),    γ -glutamyl transferase (γ-GT).  All those are important examples of the use of enzymes in clinical analysis. There are two different approaches: to determine the quantity of an enzyme or, instead, to determine a substrate of an enzyme, both, as always in Bioanalysis,, with an extremely high specificity and sensitivity. In most of the instance, the final readout is achieved with optical (visible and UV) spectrophotometry. Incoming lectures and problems will be focused on these two approaches. 

First of all, and prior to focus on enzymatic analysis,  we are going to revise the structure of the enzymes. Maybe, you are familiar with them, as biological catalyzers, with an outstanding specificity towards the substrate. 

Remember that the presentation for this topic can be found in Lesson 2. You can download it here. 

Lesson 2

Structure of biomolecules and Biorecognition. Amino Acids, Peptides and Proteins. Antibodies. Enzymes. Nucleic Acids. Biorecognition: Enzyme/Substrate. Antigen/antibody. Hybridization. Other affinity interactions in nature. Strept(avidin), Protein A and G. Aptamers.  Biomimetic recognition. The importance of water in biorecognition. Biological buffers.

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LECTURES

This picture can help you out to summarize the differences in enzyme cofactors

Lesson 5

Enzymatic analysis. Enzymes in bioanalytical chemistry. Enzymatic kinetics. Examples of reactions catalyzed by enzymes. Enzymatic inhibitors. Quantification of enzymes and their substrates. Clinical Examples. Creatinin.  Glucose.  Uric Acid. Urea. Colesterol. Aspartate aminotransferase (GOT  or  AST).  Alanine aminotransferase (ALT or GPT).     γ -glutamyl transferase (γ-GT). 

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Enzyme activity

The quantity or concentration of an enzyme can be expressed in molar amounts, as with any other chemical, but this information is in most of the cases, irrelevant since it is highly influenced by the purity, and the conditions in which it works (temperature, pH). The concentration is usually provided by in terms of activity in enzyme units.

Enzyme units

Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s⁻¹, but this is an excessively large unit. A more practical and commonly used value is international unit (IU) = 1 μmol substrate consumed per min⁻¹.

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

The enzyme unit, or international unit for enzyme (symbol IU) is the most common unit of enzyme’s catalytic activity.

International Unit

1 IU (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method.

The specified conditions will usually be the optimum conditions, which including but not limited to temperature, pH and substrate concentration, that yield the maximal substrate conversion rate for that particular enzyme. In some assay method, one usually takes a temperature of 25°C. This means that the measurement should be performed at Vmax and order 0. An increased amount of substrate will increase the rate of reaction with enzymes, however once past a certain point, the rate of reaction will level out because the amount of active sites available has stayed constant.

The enzyme unit was adopted by the International Union of Biochemistry in 1964. Since the minute is not an SI base unit of time, the enzyme unit is discouraged in favor of the katal, the unit recommended by the General Conference on Weights and Measures in 1978 and officially adopted in 1999. 

One katal is the enzyme activity that converts one mole of substrate per second under specified assay conditions, so 1 IU = 1 μmol/min = 1/60 μmol/s ≈ 16.67 nmol/s;16.67 nkat = 16.67 nmol/s. Therefore, 1 IU = 16.67 nkat, while 1 μkat corresponds to 60 IU.

Specific activity

The specific activity of an enzyme is another common unit.

This is the activity of an enzyme per milligram of total protein (expressed in μmol min⁻¹ mg⁻¹).

Specific activity gives a measurement of enzyme purity in the mixture. It is the micro moles of product formed by an enzyme in a given amount of time (minutes) under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal/kg, but a more practical unit is μmol/mg min (IU/mg⁻¹).

Specific activity is a measure of enzyme processivity (the capability of enzyme to be processed), at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme.

Turnover number 

Turnover number (also termed k_cat) is defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration  for enzymes with two or more active sites. For enzymes with a single active site, k_cat is referred to as the catalytic constant k₂. It can be calculated from the maximum reaction rate  and catalyst site concentration  as follows:.

If the molecular weight of the enzyme is known, the turnover number, or μmol product per second per μmol of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second.

Turnover numbers range from 100 up to 40 million for catalase.

Problems and exercises

Work on Your Own 

Work on this problem to learn more about enzymatic units

Crossword

Instructions. Solve the crossword, do a screenshot and post it in the forum under the topic Top five challenge!