Practical Exercises from Biochemistry Metabolism of aminoacids I.

Content of practical exercises 1. week

1. Determination of total proteins of blood serum.................................................................................1

Metabolism of proteins
The metabolism of proteins is a significant part of fundamental biochemical processes of each
cell in organism. The metabolism is composed of two main parts - proteosynthesis (formation of
proteins) and proteolysis (degradation of proteins).
To proteins undergoing metabolic processes belong proteins produced by the organism and
foreign proteins coming from food. Degradation of proteins leads to their basic building blocks -
amino acids, which can again serve, besides the other tasks, for synthesis of new proteins.
All proteins contain nitrogen (-NH2 groups), therefore their metabolism can be followed by
intake and excretion of nitrogen. The difference is called the nitrogen balance, which can be positive
- if the intake of nitrogen prevails, or negative - if the excretion of nitrogen prevails, or in
equilibrium.
The analysis of blood plasma proteins provides important data about the processes. The blood
plasma is a mixture of different proteins in the amount 65 - 85 g/L.
The knowledge of the concentrations of individual protein types in blood plasma has a
diagnostic significance. For example, the concentrations of proteins with short half-lives
(prealbumin, transferrin) are related to the level of proteosynthesis. Immunoglobulin levels inform
about the condition of the immune system. The presence of tissue enzymes in blood plasma signals
disturbances in the tissues (damage of cellular walls). Components of blood clotting system
(fibrinogen and others) provide information on bleeding or thrombosis formation.

2. Determination of total proteins of blood serum

The blood plasma consists of more proteins, which have specific functions. Usually total
proteins, albumin and globulins are determined. Albumin is the major protein of human blood
plasma. It is synthesized in the liver forming up to 50 % of all liver production of proteins. Albumin
significantly contributes to the maintenance of oncotic pressure of blood plasma. When its
concentration decreases pronouncedly then oedema occurs as a consequence of water penetration
from blood vessels into tissues.
The decrease of plasma albumin concentration can occur from these reasons:
a) decreased synthesis (chronic liver diseases, wrong nutrition and absorption disturbances)
b) abnormal distribution (hypoalbuminemia – as a consequence of increased intake of water or
increased permeability of blood capillaries)
c) abnormal excretion or degradation.

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 Practical Exercises from Biochemistry Metabolism of aminoacids I.

Specific proteins are found in blood plasma, which give valuable informations on diagnosis and
treatment determination. Changes in their concentrations are caused by trauma (injuries, surgery),
infections or cancer.
Peculiar proteins of blood plasma are enzymes appearing during some diseases (acid phosphatase,
aminotransferases, alkaline phosphatase, amylase, creatine phosphokinase, lactate dehydrogenase,
and others). They enter blood plasma after damage of tissues - namely of the heart and liver.
An important component of total proteins in blood plasma are immunoglobulins participating in the
immune defence of organism.
Besides above mentioned functions the blood plasma proteins serve as a protein pool. Two
pathophysiologic conditions can occur in humans:
Hypoproteinemia (c < 60 g/L) is characteristic for disturbances in liver proteosynthesis,
malnutrition, severe infectious diseases and severe stages of cancer.
Hyperproteinemia (c > 90 g/L) is less common. It can be caused by dehydration of organism (heavy
work or exercise, vomiting, etc.) or more seldom by increased synthesis of immunoglobulins during
chronic inflammation.

The principle of determination: The proteins react with Coomassie Brilliant Blue dye G-250
(CBB G-250) – Bradford reaction. The acidic solution of CBB G-250 is red, the dye is protonated.
After the addition of the protein to the solution, protein and dye interact together, bind to each other
and the color of the solution changes to blue because the dye molecule is deprotonated. Changing
the color of the solution can be measured as change of absorbance at a wavelength of 595 nm.

Material

Test tubes, pipettes, spectrophotometer

Coomasie Brillian Blue G-250 (CBB), blood serum, distilled water

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 Practical Exercises from Biochemistry Metabolism of aminoacids I.

Procedure
1. Pipette the solutions into three test tubes by the table:
Components (mL) Sample Standard Blank

Coomasie brilliant Blue 1.5 1.5 1.5

Serum (unknown 0.02 - -

sample)

Standard solution - 0.02 -

Distilled water - - 0.02

2. Measure absorbances of the unknown sample (Ax) and standard (As) against blank in 1 cm cell at the
wavelength of 595 nm.
3. Calculate the amount (cx) of total proteins in the unknown sample by the formula:
Ax
cx = cs .  (g/L)
As
Conclusions: Compare your value with declared one. Try to find out metabolic disturbances causing the difference of
your finding from normal values in blood serum.