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Lesson 3. COMPLEX (CONJUGATED) PROTEINS: GLYCOPROTEINS, LIPOPROTEINS, AND PHOSPHOPROTEINS. QUANTIFICATION OF PROTEINS

Questions to study.

1. Complex proteins: their definition and classification.

2. Characteristics of glycoproteins and proteoglycans: their structure, representatives and significance.

3. Characteristics of phosphoproteins: their structure, representatives and significance.

4. Characteristics of lipoproteins: their structure, representatives and significance. Blood lipoproteins.

5. Determination of proteins in the blood serum and its significance.

6. Fractionation of proteins by methods of electrophoresis and ion-exchange chromatography, and their significance in medicine.

Assignment for self-instruction

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Task

Guidelines for performing the task

1

2

3

1

Study the classification, structure and chemical properties of carbohydrates

1. Write down the formula of α-D-glucose, α-D-fructose, N-acetyl galac-tosamine, neuraminic acid

Continued of the table

1

2

3

2

Study the structure, properties and biological significance of various representatives of glycoproteins

1. Identify O- and N-glycosidic bonds between protein and carbohydrate components in molecules of glycoproteins.

2. Give examples of glycoproteins that perform the function of enzymes, hormones, blood clotting factors, transporters.

3. Characterize the structure and biological role of mucosal glycopro-teins - salivary mucin, gastromucoprotein, uroglycoproteins

3

Study the classification, structure and biological role of proteoglycans

1. Write down the classification of glycosaminoglycans. Write down the structure of disaccharide units for hyaluronic acid, chondroitin sulfate, keratin sulfate, dermatan sulfate, heparan sulfate.

2. Make a schematic drawing of proteoglycan complexes of extracellular fluid. Describe the role of hyaluronic acid in the extracellular matrix.

3. List the physico-chemical features of proteoglycan complex

4

Recall the structure of lipids

1. Describe the classification of lipids.

2. Write down the general formula of triacylglycerols (TAG), give examples of simple (triolein) and mixed (stearooleipalmitin) triacylglyc-erols. How does different fatty acid composition affect physico-chemical properties of TAG?

3. Remember the structure of phosphoglycerols - posphatidic acid, posphatidyl choline, posphatidyl ethanolamine, phosphatidyl serine.

4. What does plasmalogens mean?

5. Remember the structure of sphyngolipids - sphingomyelins, cere-brosides, gangliosides.

6. Remember the structure of cholesterol and cholesterol esters

5

Study the structure, chemical composition, methods of separation and biological role of blood serum lipoproteins

1. Make a schematic drawing of blood serum lipoprotein structure. Which methods are used to separate plasma lipoproteins?

2. What is the function of blood serum lipoproteins?

3. Fill the table to characterize the difference between chemical composition of different types of blood serum lipoproteins (see below)

6

Study the structure and function of biological membranes

1. Characterize the chemical composition of biological membranes. Make a schematic drawing of the structure of biological membranes.

2. Characterize liquid-crystal structure of biological membranes.

3. Describe the types of transmembrane transport of substances.

4. What does liposome mean? How can they be used in medicine?

7

Study the structure of phosphopro-teins

1. Characterize the bond between apoprotein and phosphoric acid residue.

2. Give examples of most common phosphoproteins and describe their significance

Ending of the table

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2

3

8

Study the methods of quantitative analysis of proteins in biological fluids

1. Formulate the principle of quantitative analysis of protein by biuret method.

2. Point out the reference level of protein in the blood and give examples of diseases associated with hypo- and hyperproteinemia

9

Study the dependence between light absorption and intensity of solution color

1. Draw the graph of dependence of light absorption on intensity color of the solution.

2. Make a schematic drawing of photocolorimeter and briefly describe the principle of protein quantification in solution

10

Study the principle of protein fraction-ation by electrophoresis

1. What does electrophoresis mean? Explain why various proteins move in electric field at a different rate. Formulate the principle of electropho-resis in protein fractionation.

2. Describe the main protein fractions of the blood.

3. Describe the significance of determining protein spectrum of the blood in medicine

11

Study the principle of protein fractionation by ion-exchange chromatography

1. What does ion-exchange chromatography mean?

2. Formulate the principle of ion-exchange chromatogramphy in protein fractionation

Fill in the table:

Type of lipoproteins

Reference level in the blood, g/l

Apoprotein,

%

TAGs,

%

Cholesterol,

%

Phospholipids,

%

(chylomicrons

VLDL

(pre-ß-lipoproteins)

LDL

(ß-lipoproteins)

HDL

(α-lipoproteins)

Library-research paper

1. Casein and its role in nutrition of newborns.

2. Methods of blood serum lipoprotein fractionation.

LABORATORY WORK

1. Determination of protein in blood serum by biuret method

When protein reacts with biuret reagent, a colored complex is formed. The coloration intensity is proportional to protein concentration in the sample.

Substances containing two and more peptide bonds enter the biuret reaction. The color intensity is directly proportional to the number of peptide bonds in solution.

Procedure

1. Take two glass tubes. Pour 0.1 ml serum in tube 1 (experimental) and 0.1 ml solution of 0.9% NaCl in tube 2 (control).

2. Add 5 ml of biuret reagent to both tubes and stir them.

3. Leave both tubes for 30 min at room temperature.

4. Measure light absorbance by photocolorimetry at 540-560 nm against the control sample.

5. Calculate the concentration of protein using calibration line. Plot of calibration line

In order to plot the calibration line, the following components should be brought to the test tubes in accordance with data in the table:

Reference standard solutions

1

2

3

4

5

Protein content (mg)

20

40

60

80

100

Protein concentration (g/l)

20

40

60

80

100

Analytical standard solution of protein (ml)

0.2

0.4

0.6

0.8

1.0

Water (ml)

0.8

0.6

0.4

0.2

0

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