Biochemistry laboratory work (Практикум по биохимии). Методическое пособие

МИНИСТЕРСТВО НАУКИ И ВЫСШЕГО ОБРАЗОВАНИЯ РФ НОВОСИБИРСКИЙ ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ Институт медицины и психологии В. Зельмана

А. А. Шефер, С. Н. Тамкович

BIOCHEMISTRY LABORATORY WORK

Практикум по биохимии

Методическое пособие

Новосибирск

2023

3

4

Laboratory work №1: Determination of the activity of enzymes based on the example

of determination of lactate dehydrogenase activity in blood plasma

Enzymes are proteins that are produced by living cells and have the ability to catalyze various chemical compounds.

A characteristic of the activity of enzymes is the rate at which they catalyze a particular reaction. It is measured by the rate of transformation of the substrate or the rate of accumulation of reaction products. Measurement is the initial rate of conversion, never the amount of substrate converted during a certain period of time. The activity unit (E) is the amount of an enzyme that catalyzes the conversion of one micromole of a substrate in min under standard conditions

The following methods are used to determine the enzymatic activity:

1.Chemical method — quantification of substrate or products using chemical reagents (O-glycosylhydrolase - by formation of reducing sugars).

2.Spectrophotometric method — measuring the rate of enzymatic reaction by the change in absorbance of the substrate at a characteristic wavelength (lyase - by formation of double bonds).

3.Manometric method — determining the amount of gas released during the reaction (oxidases - by absorption of O2, decarboxylases - by emission of CO2).

4.Polarimetric method — change in optical rotation is recorded (β-fructofuranosidase).

5.Chromatographic — quantitative determination of substrate or products using different types of chromatography: paper (analysis of sugars), thin-layer (glycosides with complex aglycones), HPLC (amino acid analysis, etc.).

5

The objective of the work is to determine the activity of LDH in blood serum using a spectrophotometric method.

Lactate dehydrogenase (LDH) is an intracellular zinc-containing glycolytic enzyme involved in the reversible conversion of lactate to pyruvate and found in most body tissues. LDH is active mostly in skeletal muscle, heart, kidneys, liver, and erythrocytes.

Figure 1. Reaction catalyzed by LDH

Five different forms (isoenzymes) of LDH exist. They differ in their molecular structure and location in the body. Whichever of the five predominates determines the main mode of glucose oxidation — aerobic (to CO2 and H2O) or anaerobic (to lactic acid). Such distinction is caused by the different affinity of one or another isoenzyme to pyruvic acid. For myocardium and brain tissue, LDH-1 is the major one; for erythrocytes, platelets, and kidney tissue, LDH-1 and LDH-2 are dominant. In lungs, spleen, thyroid, pancreas, adrenal glands, and lymphocytes, LDH-3 predominates. LDH-4 is found in all tissues with LDH-3, as well as in granulocytes, placenta, and male germ cells, which also contain LDH-5. Isoenzymatic activity in skeletal muscle (in descending order): LDG-5, LDG-4, LDG-3. The LDH-5 isoenzyme is the most common in the liver, while LDH-4 has less activity. In normal blood serum, all fractions of the enzyme are determined with low activity as part of the total index — total

6

LDH. Their activity in blood is distributed as follows: LDG-2 > LDG-1 > LDG-3 > LDG-4 > LDG-5.

In the case of diseases accompanied by tissue damage and cell destruction, LDH activity in the blood increases. Therefore, it is an important marker of tissue destruction. Although increased enzyme activity does not specify any particular disease, its determination in complex with other laboratory tests helps in the diagnosis of pulmonary infarction, muscular dystrophy, and hemolytic anemia. Elevated LDH activity can be detected in newborns, pregnant women, and after intense physical exertion.

In patients with angina pectoris (stenocardia) the enzyme activity remains unchanged, but in case of myocardial infarction it begins to increase within 8-10 hours with a maximum in the first 24-48 hours after the heart attack and returns to the norm after 10-12 days. Increased LDH with normal AST activity 1-2 days after chest pain indicates pulmonary infarction.

In the distinguishing diagnosis of myopathies, this analysis can help to clarify the pathophysiological mechanisms of the disease. Thus, in muscle dysfunction associated with neurogenic diseases, LDH does not increase, but in muscle damage due to endocrine and metabolic pathologies, LDH activity increases.

LDH activity in blood can increase as a result of many malignant neoplasms, it decreases with effective treatment, which sometimes is used for dynamic monitoring of cancer patients.

What is the study used for?

•For diagnosis of acute or chronic tissue damage in a comprehensive examination of a patient.

•For distinguishing diseases in case of acute chest pain (myocardial infarction, angina pectoris, pulmonary infarction).

•To detect diseases accompanied by erythrocyte hemolysis.

•For monitoring the course of cancer in therapy.

•To check liver and kidney abnormalities.

•To diagnose muscle tissue lesions.

7

When the study is necessary?

•In case of suspected acute or chronic tissue and cellular damage in the body.

•In a comprehensive preventive examination of a patient.

•When the course of some chronic diseases (muscular dystrophy, hemolytic anemia, liver and kidney diseases), or cancer pathology is monitored.

Equipment and materials:

1)spectrophotometer,

2)tips for pipettes,

3)automated pipettes of variable volume for 20-100 and 100-1000

µl,

4)rubber gloves,

5)test tubes of 5-10 ml capacity,

6)thermostat,

7)stopwatch,

8)cotton swabs,

9)EDTA Vacutainer K3,

10)disposable needles for blood sampling,

11)needle adapter-holder.

Reagents:

1)reagent 1 of the set LDG-UV-Novo JSC "Vector-best",

2)reagent 2 of the set LDG-UV-Novo "Vector-best" JSC,

3)0.9% NaCl,

4)distilled water,

5)ethanol 96%.

Work plan

Attention! Work must be performed with single-use gloves.

1. Wipe the workplace, pipettes and gloves with a cotton swab moistened with 70% ethanol.

8

2. Draw blood by venipuncture up to a risk of 9 ml.

3. After gentle agitation centrifuge tube with blood for 10 min at 3000g.

4. Collect plasma in a new tube.

5. Dissolve the contents of reagent vial 2 in reagent vial 1 and mix by hand.

6. Heat the reagent mixture to 37°C for 15 min.

7. Add 25 µl of test plasma sample to 1000 µl of heated solution using an automatic pipette, mix well and start the stopwatch.

8. Calibrate the spectrophotometer against air.

9. After 30 sec of sample mixing measure the optical density at 340 nm.

10. Repeat the measurements at 1 min intervals.

11. Calculate the change in optical density using the formula

∆ = ∆ .

Here ∆En is the measurement of the optical density of the sample, t is the interval between measurements LDH activity (A) is

Here V — final volume of reagents in the cuvette, v — volume of analyzed sample,

9

ε — molar extinction coefficient NADH (6,22l/(mol×cm),

1000 — conversion coefficient for the expression of total LDH activity.

Laboratory work №2: Determination of hormones in the blood by ELISA, by example TSH level determination

The enzyme immunoassay (ELISA) is a method based on the reaction of an antigen with an antibody to determine the concentration of one of these reagents in a solution. The high stability of reagents, simplicity of registration, and many other advantages of the ELISA method have contributed to its broad adoption in various fields of medicine and science, agriculture biological industry and environmental protection. The objective of the work is quantitative determination of TSH level using enzyme immunoassay.

In the immunoassay, the insertion of an enzyme tag is carried out by covalent binding of an enzyme molecule to an antigen or antibody molecule. The most accessible for binding is the ε-amino group of lysine, the carboxyl groups of asparagine cysteine residue of the surface hydrophilic side chains of the protein. Obviously, this modification should not affect the functional groups of the active center of the enzyme and should not affect the on the catalytically active conformation of the whole enzyme

For the registration of the enzyme activity in ELISA, the most common method is the photometric method. Substrates used for enzymes are substances whose transformation products are colored, or, alternatively, the products of enzymatic transformation are chaining its color. Coloured compounds absorb visible light (wavelengths of 400-700 nm), and the optical density of the solution in a certain range is directly proportional to the concentration of the substance (according to Bouguer — Lambert — Ber law).

10