Proteins are high molecular weight organic compounds of amino
acids that are combined together by peptide bonds to form polypeptide chains.
All proteins contain carbon, hydrogen, oxygen, nitrogen and sulfur. Proteins
may also contain phosphorous, iodine, copper, zinc and other elements. The
presence of nitrogen makes protein distinct from carbohydrates and lipids. The
average nitrogen content is approximately 16%.
Chemical structure
Alpha amino acids constitute that class of organic acid which
contains an amino group located on the carbon atom adjacent to the group.
The R group represents the remainder of the molecule and varies
from H in glycerine to the complex indole ring in tryptophane.
Amino acids contain both the acidic carboxyl group (protein
donating) and the basic amino group (proton accepting) within the molecules.
These compounds are referred to as amphoteric compounds and behave both as acid
and bases. At about normal pH, the carboxyl group is dissociated and the NH2
group has bound the protons. These types of ionized molecules with negative and
positive charges are referred to as zwitterion or ampholyte. Isoelectric point
refers to the pH value at which the sum of molecule charges equals zero.
General function
1.
To maintain osmotic pressure in the blood
(albumin accounts for 75% of the colloid osmotic pressure of plasma).
2.
To act as reserve of protein for tissue,
repair and growth.
3.
To act as pH buffers (plasma proteins but
especially hemoglobin).
4.
To provide factors necessary for normal blood
coagulation.
e.g. fibrinogen, prothrombin,
antihemophilic factor, factor V and VII, plasma thromboplastin component (PTC),
plasma thromboplastic antecedent (PTA), etc.
5.
To provide necessary enzymes (biochemical
catalyst) in the blood.
e.g. proteinase, peptidase,
amylase, etc.
Metabolism of protein
1.
Digestion of protein begins in the stomach
immediately after ingestion. Gastric secretion includes hydrochloric acid and
pepsin.
a.
Hydrochloric acid denatures the protein,
unfolding them as the bonds that form the secondary, tertiary and quaternary
structures are broken.
b.
Pepsin acts specifically on the peptide bonds
between those amino acids containing an aromatic ring or carboxylic acid in
their R group, breaking the proteins into shorter polypeptides. As the
polypeptide move into small intestine, the pH changes from acidic to basic and
pepsin in inactivated.
2.
The pancreas secretes the zymogens (i.e.,
inactive precursors or proenzymes): trypsinogen, chymotrypsinogen, proelastase
and procarboxypeptidase into the small intestines where they are converted to
their more active forms.
a.
Trypsin acts on the peptide bonds between the
amino acids with basic R group.
b.
Chymotrypsin breaks the peptide bonds between
amino acids with neutral R groups while elastase acts on bonds between the
small amino acids such as glycine, alanine and serine.
c.
Carboxypeptidase attacks the carboxyl
terminal amino acid, liberating amino acids one at a time.
d.
Aminopeptidase secreted by the small
intestine acts on the amino terminal end to force single amino acids.
3.
Digestion is completed as free amino acids
are absorbed across the intestinal wall, a process that requires active
transport and is energy dependent.
4.
Albumin, alpha and beta globulins,
prothrombin and fibrinogen are all formed in the liver. Gamma globulins are
formed in the liver and in all reticuloendothelial tissues of the body.
5.
A process known as protein turnover takes
place in the body wherein proteins are degraded and resynthesized to be
distributed in other parts of the body.
6.
The end product of protein catabolism are:
a. Urea
b. Carbon
monoxide
c. Water
d. Uric acid
e. Phosphates
f.
Creatinine
7.
Growth hormone and insulin increases protein
synthesis. Glucocorticoids and thyroid hormone increase protein catabolism.
Classification of protein
A.
According to levels of structure
1.
Primary structure – determined by which amino
acids are present, their sequence and the number of amino acids. One amino acid
substitution can alter biologic activity, even in a large protein.
2.
Secondary structure – is the shape the strand
of amino acids takes as amino acids interact with adjacent amino acids through
hydrogen bonds, disulfide disulfide linkages between the cysteine amino acids
and other polar and nonpolar R group interactions. The peptide bond does not
rotate much, but other bonds are free to rotate. This rotation allows
conformational changes that cause the secondary shape to form. The shape maybe
described:
a. Alpha –
helix – coil or ring
b. Beta pleated
sheath – flat or corrugated
c. No apparent
pattern – random
3.
Tertiary structure – is the three–dimensional
structure that forms as the amino acids interact with more distant members of
the chain causing the chain to fold and takes its characteristic shape.
4.
Quaternary structure – is formed when two or
more chains are united. These chains are called monomers or subunits and the
final proteins formed are called dimers, tetramers or oligomers.
Denaturation or inactivation – is
the disruption of the bonds holding the secondary, tertiary or quaternary
structure. This is accomplished by:
1.
Heat
2.
pH changes
3.
Chemicals (e.g., detergents, metals,
solvents)
4.
Mechanical factors
·
In the clinical laboratory setting, it is
important to note that excessive heat, a freeze –thaw cycle or vigorous mixing
can break these bonds and thus denature protein. An enzyme can lose its
activity, a receptor can lose its ability to bind, and an antigen can lose its
antigenicity and fail to be recognized by the antibody if these proteins are
denatured before being assayed.
B.
According to shape (defined by
comparing the ratio of length to breath)
1.
Globular proteins have a ratio of less than
10. It has length to breath ratio closer to 2 or 3.
e.g. hormones, enzymes, hemoproteins
2.
Fibrous proteins have a ratio greater than
10.
e.g. myosin, keratin
C.
According to solubility
1.
Albumin – these proteins that are soluble in
water and soluble in dilute and concentrated salt solutions but insoluble in
highly concentrated salt solutions such as saturated ammonium sulfate.
2.
Globulin – these are proteins that are
insoluble in water, soluble in weak neutral salt solutions but insoluble in
concentrated salt solutions.
3.
Albuminoids – a special group of proteins
characterized by being essentially insoluble in some common reagents.
e.g. collagen, elastin, keratin
D.
According to composition
1.
Simple proteins – composed of amino acids
only.
2.
Conjugated proteins – consists of amino acid
chains and non–amino acid molecules. The amino acid portion of the protein
molecule is called the apoprotein. The non– amino acid portion is called
the prostethic group.
a. Chromoprotein
– contain an organic prosthetic group that is linked to some metal ions.
e.g. Myoglobin, hemoglobin
b. Metalloprotein
– some metal ions are directly attached to the protein.
e.g. Ferritin, ceruloplasmin
c. Lipoprotein
– contain bound cholesterol, phospholipid or both.
d. Glycoprotein
– contain complex carbohydrate in their structure
e.g. Mucin and crossomucoid
e. Nucleoproteins
– protein is associated with chains of DNA.
E.
According to specific function
1.
Catalyst (enzymes)
2.
Regulatory proteins, including receptors,
hormones, repressor and inhibitors
3.
Transport proteins
4.
Structural proteins including such
subspecialties as contractile, fibrous and keratinous proteins.
5.
Protective proteins, including
immunoglobulins and complement
6.
Oncofetal and placental proteins
7.
Proteins that have unknown functions
Laboratory techniques used in protein studies
1.
Salt or solvent fractionation
Salt fractionation will
differentiate proteins according to their solubility using different
concentration of salt solutions. The most frequently used salt is ammonium
sulfate.
2.
Ultracentrifugation
Because proteins are made up of
different numbers and kinds of amino acids, individual proteins differ in
molecular weight. If a protein solution is placed in a centrifuge operating at
a very high speed, the high centrifugal force will force the heavier molecules
to sediment or settle out faster than lighter molecules. This is accomplished
by the use of an ultracentrifuge. This technique is particularly more useful in
separating light lipoproteins from other heavier serum proteins.
3.
Chromatographic separations
Because of differences in size,
shape and chemical composition, different proteins will be absorbed on and can
be eluted from various adsorbents at different rates. By control of pH, buffer
type and concentration and proper choice of adsorbents and eluting solutions,
individual proteins can be separated from each other and obtained in a highly
purified form.
4.
Immunodiffusion
5.
Electrophoresis
6.
Capillary electrophoresis
7.
Immunoelectrophoresis
8.
Isoelectric focusing
10. Two–dimensional
electrophoresis
(Discussion of #4 – 10 can be
found here)
****** PROTEIN FRACTIONS ******
1.
Albumin 55%
2.
Globulin 38%
a.
Alpha–1 globulin and 14%
Alpha–2 globulin
b.
Beta globulin 13%
c.
Gamma globulin 11%
3.
Fibrinogen 7%
Total Protein 100%
****** THE TOTAL PROTEIN ******
Methods for total protein determination
1.
Gravimeteric method
Protein is precipitated by a
protein precipitant and the precipitated is washed, dried and weighed. This
method requires large amounts of serum and its time consuming.
2.
Based on the influence of protein
on specific gravity
a.
Falling drop method
b.
Copper sulfate specific gravity method
3.
Kjeldahl method
A standard reference method based
on nitrogen determination, one gram of nitrogen is equivalent to 6.54 grams of
protein. The basic procedure involves the following steps:
a.
Precipitate the protein with acid
b.
Wash the precipitate to remove non–protein nitrogen
c.
Oxidize the protein with H2SO4
and heat in the presence of catalyst to produce NH4SO4
and other end products.
d.
Boil to remove end products (CO2,
CO, H2O and SO2)
e.
Add excess OH– and distill NH3
into boric acid
f.
Titrate distillate to determine NH3
content
4.
Phenol method (Lowry method)
This method employs Folin and
Ciocalteau reagent (lithium salts) of phosphotungstic–phosphomolybdic acid in
an alkaline solution and are reduced to a blue color in the presence of
tyrosine and tryptophan. This is known as the phenol method because phenol is
used as oxidizing agent.
5.
Turbidimetric method
Diluted serum is treated with
sulfosalicylic acid resulting in the precipitation of protein. The resultant
precipitate of protein is read photometrically as a turbidity.
6.
Measurement of refractive index
The refractive index of an
aqueous solution increases as the protein concentration increases and therefore
RI measurements can be used to determine the protein content.
7.
Ultraviolet light absorption
8.
Biuret method / Kingsley et.al.
All proteins contain a large
number of peptide bonds. When a solution of protein is treated with a Cu+
ions in moderately alkaline media, a colored chocolate complex of unknown
composition is found between the Cu2+ ion and the carboxyl (=CO=)
and the (=N=H) of the peptide bonds. An analogous reaction takes place between
the cupric ions and the organic compound, biuret and therefore the reaction is
called Biuret reaction.
The intensity of color produced
is proportional to the number of protein bonds undergoing reaction, thus to
quantity of protein in the sample. The proteins are separated by precipitating
the globulins by the addition of concentrated sulfate–sulfite solution,
determining the albumin in the remaining solution and calculating the globulin
by the difference.
A rapid, simple and accurate
method for protein determination. It is based on the formation of complex
colored compound from biuret linkage present in protein.
Biuret reaction is a reaction
between cupric ions in alkaline solution with substances containing two or more
peptide bonds to produce a violet color or reddish purple color.
Reagents:
a.
22.6% Na2SO4 – to
precipitate globulins
b.
Sulfuric ether – to lower the density of the
precipitated globulin and to facilitate the separation of globulin by
centrifugation.
c.
Biuret reagent – color developer
Composition of Biuret reagent
(1) Alkaline
copper sulfate
(2) Rochelle
salt (Na K tartrate)
(3) Sodium
hydroxide
(4) Potassium
iodide
Computations:
Total protein – albumin = globulin
Albumin ÷ Globulin = A/G
ratio
Important notes
a.
Never use crystallized Na2SO4
as this will cause incomplete precipitation of globulin.
b.
Do not shake the serum–salt mixture
vigorously to prevent denaturation of albumin
c.
In pipetting the albumin fraction, care must
be observed so as not to disturb the precipitate and wipe the pipet in
transferring the solution to remove any adhering globulin.
1921 – Howe recommended the use
22% (w/w) sodium to precipitate globulin
1940 – Kingsley introduced the
use of ether as a means of separating the precipitated globulin from the
soluble albumin without resorting to the slow tedious filtration.
Interferences
a.
Falsely elevated: (1) Lipemia (2) Hyperbilirubinemia (3) Hemolysis
b.
Falsely low: (1)
High blood ammonia
Clinical significance
a.
Decrease in serum total protein is seen in
(1) Malnutrition
/ Thiamin deficiency
(2) Glomerulonephritis
– disorder characterized by an inability of kidney to retain large molecular
weight protein
(3) Hemorrhage
(4) Liver damage
b.
Increase serum total protein is seen in
(1) Hemoconcentration
(2) Infectious
hepatitis
c.
Inverted A/G ratio: Multiple myeloma
****** ALBUMIN
******
Method of quantitating albumin
1.
Dye Binding Method using
a.
BCG – Bromcresol green
b.
HABA–2–(4’–hydroxybenzene)
Interfering substances
HABA BCG
Hemolysis increased slight increase
Bilirubin decrease decrease
Salicylate slight decrease no effect
Heparin increase decrease
2.
Turbidimetry and Nephelometry
3.
Immunoassay with labelled
antibody
4.
Immunofixation used in
combination with electrophoretic techniques
Clinical significance of albumin values
Total protein and albumin in disease state
****** PRESENCE OF PROTEIN IN OTHER BODY FLUID ******
1.
Urine
Protein is not normally present
in urine. Protein present in urine is generally albumin and if persistent,
indicates kidney disease.
Method of quantitation
a.
Turbidimetric method using sulfosalicylic
acid
b.
Qualitative dipstick method
c.
Quantitative method
2.
Cerebrospinal fluid
Normal value of CSF protein is
usually 15–45 mg / dl or less. It is elevated in multiple sclerosis.
Method of quantitation
a.
Turbidimetric
b.
Coomasie blue dye
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