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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5709_Библиотеки_им_академика_М_И_Перельмана.pdf
122
Pharmaceutical Chemistry of Natural Products
NH CHCONHCHCONH
2
⫹
CHCOOH
NH CHCONHCHCONHCHCH OH
22
LiAlH
4
R
1
R
2
R
3
R
1
R
2
R
3
Hydrolysis
NH CHCH OH
22
R
3
⫹
NH CHCONHCHCOOH
2
R
1
R
2
4. Enzymatic method: The enzyme carboxyl peptidase attacks peptides (or proteins) only at the
carboxyl end of the peptide. After hydrolysis, the amino acids are characterized and hence
their sequence can be established.
PROTEINS
Proteins are probably the most important class of biochemical molecules, in addition to lipids and
carbohydrates which are also essential for life. Proteins form the basis for the major structural
components of both animal and human tissues. They regulate a variety of activities in all known
organisms, from replication of the genetic code to transporting oxygen, and are generally responsible
for regulating the cellular machinery and determining the phenotype of an organism. Proteins are
natural polymer molecules consisting of amino acid units. The number of amino acids in proteins
may range from two to several thousands.
Biological Functions
Proteins possess diverse biological functions as discussed below:
1. Some proteins such as collagen in connective tissue and keratin in hair function as biological
structural materials.
2. Antibodies providing immunity against diseases are formed from blood proteins.
3. Hormones are proteins which regulate various metabolic processes (e.g. insulin maintains
blood sugar level).
4. Enzymes which catalyses the biological reactions are also made up of proteins.
5. During cell division, genetic message is transmitted through nucleoproteins which are the
important constituent of genes.
6. In haemoglobin, the protein present in it acts as oxygen carrier.
Classification of Proteins
Proteins are classifi ed into simple, conjugated and derived proteins on the basis of increasing
complexity in their structure.
Amino acids, Peptides and Proteins
123
1. Simple proteins: These proteins on hydrolysis yield only α-amino acids. They are further
classifi ed into following classes on the basis of the decreasing solubility.
(a) Albumins: They are soluble in water, acids and alkalies and are coagulated by heat and
precipitated by saturated solution of ammonium sulphate. These are usually defi cient in
glycine. Examples are serum albumin (blood), ovalbumin (egg white) and lactalbumin
(milk).
(b) Globulins: They are insoluble in water, but soluble in dilute solutions of salt, strong inorganic
acids and alkalies. They are coagulated by heat and precipitated by half-saturating their
solutions with ammonium sulphate. Generally, globulins contain glycine. Examples are
serum globulin (blood), myosin (muscle), vegetable globulin and tissue globulin.
(c) Prolamins: They are insoluble in water or salt solutions, but soluble in dilute acids, alkalis
and 70–90% ethyl alcohol. They contain large amounts of proline and are defi cient in
lysine. Examples are gliadin (wheat and rye), zein (corn), secalin (rye) and hordein
(barley).
(d) Glutelins: They are insoluble in water and dilute salt solution, but soluble in dilute acids
and alkalis. They are coagulated by heat and are comparatively rich in arginine, proline
and glutamic acid. Examples are glutenin (wheat) and oryzenin (rice).
(e) Scleroproteins (albuminoids): They are insoluble in water and salt solution, but soluble in
concentrated acids and alkalis. They are attacked by enzymes. Common examples are
keratin (from hair, nails, hooves, horns and feathers) and fi broin (from silk). Albuminoids
are subdivided into two types.
(i) Collagen: It is the most common protein in the mammalian body. It is found in the
skin, tendons and bones. They are attacked by pepsin and trypsin. When boiled with
water, they form a water-soluble protein (gelatin).
(ii) Elastin: They are found in the tendon, arteries and other elastic tissues. They are
attacked slowly by trypsin.
(f) Histones: Naturally, they are complex chemicals, soluble in water or dilute acids but
insoluble in dilute ammonia. They are not coagulated by heat. These are hydrolysed by
pepsin and trypsin. Proteins of the nucleic acids and haemoglobin are the examples.
(g) Protamines: They are soluble in water, dilute acids and dilute ammonia. These are more
basic than the histones and have a simpler structure and found in sperm cells of certain
fi sh. Examples are sturine (sturgeon), scombrine (mackerel), salmine (salmon) and
clapeine (herring).
2. Conjugated proteins: Upon hydrolysis these proteins yield α-amino acids and nonproteinous
substances. They contain simple protein molecules linked with a nonprotein substance. The
nonproteinous moiety is referred as the prosthetic group. On the basis of the prosthetic group
present in it, they are subdivided as follows:
(a) Glycoproteins (mucoproteins): They are produced by adding sugar residues through
o-glycoside linkages to the hydroxyls of serine and threonine residues or via N-glycoside
linkages to the amino of asparagine. The prosthetic group present here is a carbohydrate
or a derivative of carbohydrate (e.g. mucin of saliva).
(b) Phosphoproteins: The prosthetic group present here is phosphoric acid (e.g. casein of
milk).
(c) Lipoproteins: They have the phosphorylated hydroxyl groups of serine or threonine. The
prosthetic group present here is phospholipid (a class of fat). An example of lipoproteins
is serum.
(d) Nucleoproteins: The prosthetic group present here is a nucleic acid (e.g. nuclein).
124
Pharmaceutical Chemistry of Natural Products
(e) Chromoproteins: They contain coloured prosthetic group due to the presence of a metal in
their structure. The most common example is haemoglobin in which haemin is prosthetic
group and globin is the protein; another example is chlorophyll.
3. Derived proteins: They are the products obtained by the action of heat, chemical or enzymatic
agents on natural proteins. The derived proteins (intermediate hydrolysis products) are
further classifi ed on the basis of progressive cleavage into denatured proteins, primary
proteoses (metaproteins), secondary proteoses, peptones, polypeptides and simple peptides.
(a) Denatured proteins: They are insoluble proteins formed by the action of heat on proteins.
(b) Primary proteoses (metaproteins): They are insoluble in water, dilute salt solution and soluble
in acids or alkalis. They are precipitated by half-saturation with ammonium sulphate.
(c) Secondary proteoses: They are soluble in water and are coagulated by heat and are precipitated
by saturation with ammonium sulphate.
(d) Peptones, polypeptides and simple peptides: They are soluble in water. They are neither
coagulated by heat nor precipitated by saturation with ammonium sulphate.
4. Proteins may also be classifi ed according to their molecular structure and behaviour. The
classifi cation is as follows:
(a) Fibrous proteins: They are formed by the condensation of (neutral amino acids) monoam-
ino monocarboxylic acids and there is a few or no branching. The linear protein chains
are held together by intermolecular hydrogen bonds. These proteins are insoluble and
strong, and therefore play a structural role in organisms for support or protection. Table
3.1 shows some examples of fi brous proteins and their source(s):
(b) Globular proteins (spheroproteins): They are more highly branched and cross-linked
condensation products of basic or acidic amino acids. As the globular proteins have
diamino and dicarboxylic acids, branching and cross-linking takes place by the usual
peptide linkage involving the second amino group of one amino acid and the second
carboxyl group of the another amino acid. The peptide chain is stabilized by intramolecular
hydrogen bonds (e.g. casein of milk and globulin of haemoglobin).
Table 3.1 Fibrous proteins and their sources
Fibrous proteins Source
Keratins Hair, fi ngernails and bird feathers
Collagens (the most abundant protein in a vertebrate body) Connective tissues such as cartilage
Elastins Ligaments, around blood vessels
Qualitative Chemical Tests for Proteins
On treatment with some specifi c reagents proteins give characteristic colour. So these reactions
are used for the identifi cation of proteins. These tests are specifi c for some groups present in the
protein molecule and therefore all proteins will not answer for all the tests.
1. Biuret test: With 10% sodium hydroxide solution, the protein is warmed and then a drop of
dilute copper sulphate solution is added. Formation of reddish violet colour indicates the
presence of the grouping, –CO–NH–CHR–CO–NH– (a dipeptide linkage).
Alkaline solution of proteins + Dilute solution of CuSo
4
Red or violet colour
Amino acids, Peptides and Proteins
125
The test is given by all proteins, peptones and peptides except dipeptides which do not
contain two peptide linkages. This test name is derived from the fact that the test is also
positive for the compound biuret, H
2
N–CONHCONH
2
, formed from two molecules of urea
by heating.
CO C
H
N
R
1
CO CHR
N
N
Cu
N
R—CH
CO
CH CO⭈
R
2⫺
2Na
⫹
2. Xanthoprotein reaction: Certain proteins on treatment with concentrated nitric acid give yellow
colour which becomes orange on the addition of alkali. Proteins having at least 1 mole of
aromatic amino acid such as tryptophan and tyrosine answer this test.
3. Millon’s reaction: When proteins are treated with Millon’s reagent (mercuric nitrate in nitric
acid containing traces of nitric acid), a white precipitate is obtained which upon heating gives
red colour.
Heating
Proteins + Millon’s reagent White precipitate Red colour
4. Test for proteins containing sulphur: Mix 5 mL test solution with 2 mL of 40% NaOH and
two drops of 10% lead acetate solution, and boil the solution. The solution turns black or
brownish due to the formation of lead sulphide (PbS).
5. Precipitation test: The test solution gives white colloidal precipitate with the following reagents:
(i) absolute alcohol, (ii) 5% mercuric chloride
solution, (iii) 5% copper sulphate solution,
(iv) 5% lead acetate, (v) 5% ammonium sulphate.
6. Ninhydrin reaction (indane-1,2,3-trione hydrate): The colours formed with amino acids, peptides
and proteins are different in this test. Compounds such as ammonia, ammonium salts and
certain amines also answer this test. Amines (including α-amino acids) react with ninhydrin
to give a coloured product. It can be used qualitatively (e.g. for chromatographic visualization)
or quantitatively (e.g. for peptide sequencing). The α-amino acids typically give a blue–purple
product. Proline, a secondary amine, gives a yellow–orange product. The test is sensitive
enough that ninhydrin can be used for the visualization of fi ngerprints.
O
O
OH
OH
R
O
⫺
O
N
O
O
C
O
R H
NH
2
CHCOOH
OH
⫺
⫹⫹
CO
2
⫹
4 H O
2
2
Structure of Proteins
Proteins are polymers of amino acids linked by peptide bonds. In order to establish the structure
of a protein, the following points should be addressed:
126
Pharmaceutical Chemistry of Natural Products
N
H
O
C
C
C
O
N
H
H
N
C
C
O
N
C
C
O
H
C
N
H
O
C
C
C
O
N
H
H
N
C
C
O
N
C
C
O
H
C
N
H
C
C
O
C
C
O
N
C
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
OHO
C
HH
N
Quaternary
structure
Tertiary
structure
 Pleated sheet
Alpha helix
Primary
structure
Primary structure
Secondary structure
1. The nature of amino acids.
2. The number of such particular amino acids present in one molecule of the protein.
Amino acids, Peptides and Proteins
127
3. Sequence in which the different amino acids are arranged in a protein.
Note: Points ‘1–3’ constitute the primary structure of protein.
4. Shape of the peptide chain (linear, cyclic, branched or arranged in the form of helix).
Note: Point ‘4’ constitutes the secondary structure of protein.
5. The forces responsible for holding the peptide together.
6. The mode in which individual peptide chains are arranged in defi nite manner to a
macromolecule of an individual shape (folded, refolded).
Note: Points ‘5’ and ‘6’ constitute the tertiary structure of protein.
7. The number of peptide chains and their arrangement in the natural proteins.
Note: Point ‘7’ constitutes the quaternary structure of protein.
Primary structure of proteins
Primary structure is simply the sequence of residues making up the protein. Thus, the primary
structure involves only the covalent bonds that link the residues together. The minimum size of
a protein is defi ned as about 50 residues—smaller chains are simply called peptides. Therefore, the
primary structure of a small protein would consist of a sequence of 50 or more residues. Even such
small proteins contain hundreds of atoms and have molecular weights of over 5000 dalton (Da).
There is no theoretical maximum size, but the largest protein discovered so far has about 30,000
residues. Because the average molecular weight of a residue is about 110 Da, the single chain has
a molecular weight of over 3 million dalton.
HN
3
⫹
Gly
Ile
Yal
Cys
Glu
Gln
Ala
Ser
Leu
Asp
Arg
Cys
Yal
Pro
Lys
Phe
Tyr
Thr
Leu
His
Lys
Asn
COO
⫺
The following stages are involved in the determination of complete primary structure.
1. Purifi cation: Near its isoelectric point, the crude sample of given protein is precipitated out.
The sample is prepared free from salts by dialysis or gel fi ltration and then fractionated by
chromatographic methods. The purifi ed sample is isolated by freeze drying, its purity is then
assessed by electrophoresis, countercurrent distribution and ultracentrifuge studies which
also determine the molecular weight of the protein. The nonprotein materials are removed by
extraction, while insoluble proteins are much more easily purifi ed. The structure of insoluble
proteins has been determined mainly using X-ray studies on the native material.
2. Determination of amino acids composition: The protein is hydrolysed by means of 6N HCl at
110°C or enzyme (peptidase) to individual amino acids. Various amino acids are separated
and analysed by a number of methods. The commonly used method being ion-exchange
chromatography.
128
Pharmaceutical Chemistry of Natural Products
Ion-exchange chromatography: This chromatographic column is packed with sulphonated
polystyrene divinyl resin which is having highly polar SO
3
H groups. On the column of the
resin, amino acid solution is placed. The amino acid is converted into ammonium ion by the
transfer of a proton and the resin into sulphonate anions. On the basis of relative acidity
of the acid form of the amino acid, ammonium ions of different amino acids are adsorbed
on the surface of the sulphonic acid resin anion. With a buffer solution of specifi c pH, the
column is eluted to separate the amino acids which are isolated and weighed or reacted with
ninhydrin reagent and then estimated spectrometrically.
Electrophoresis: The chemical properties of the naturally occurring amino acid and its
C
14
-labelled variety which is added from the outside are the same, and therefore the two can
be isolated together. The amount of the naturally occurring form can be easily determined by
measuring the level of radioactivity. Using biological assays the amount of each amino acids
present in the hydrolysate can also be determined.
pH-Dependent precipitation: Amino acids have different isoelectric points with different
solubilities and hence they may be separated from each other. The pH of the peptide or
protein hydrolysate is changed gradually in order to successively precipitate amino acids at
their respective isoelectric points.
3. End-group determination: The C-terminal and N-terminal amino acids of a polypeptide or
protein can be determined by various methods (see page 118).
4. Disulphide bond cleavage: Prior to sequence determination, the disulphide bond, if present in
the protein molecule, must be broken. This is achieved by oxidation using performic acid or
reduction using sodium borohydride.
CH SH
2
⫹
CH SH
2
NaBH
4
CH
2
S
S
CH
2
HCOOOH
⫹
CH SO
23
⫺
CH SO
23
⫺
Cysteic acid residues
5. Determination of sequence of amino acids: By end-group determination the polypeptide chains
obtained after disulphide bond cleavage are separated and characterized. By adopting Edman’s
method, the sequence of amino acids in the chain is then determined. It is possible to remove
the amino acids in a stepwise manner from a chain containing as much as 60 amino acid
residues.
6. Determination of the position of the disulphide bonds: With the help of enzymes the original
molecule is degraded to form peptides having disulphide bonds. Sequence determination on
these peptides reveals sequences which coincide with those already found in the constituent
chains around the cysteine residues.
The primary structure (i.e. exact sequences of amino acids) of proteins has important biological
implications. For example, in the amino acid chain of haemoglobin, at one point, if a glutamic acid
is replaced by a valine, a different haemoglobin called haemoglobin S is obtained. This haemoglobin
S causes sickle cell anaemia.
Amino acids, Peptides and Proteins
129
Secondary structure of proteins
Peptide chain conformation in protein molecule is dealt with secondary structure. This level
of structure describes the local folding pattern of the polypeptide backbone and is stabilized by
hydrogen bonds between N–H and C=O groups. All polypeptide chains are not capable of forming
the α-helix; the amount of the helix form varies in proteins from 0 to about 100%. X-ray studies of
peptides indicated that the entire amide group is fl at. Carbonyl carbon, nitrogen and the four atoms
attached to them all lie in one plane. Various types of secondary structure have been discovered,
but by far the most commonly referred to structure is the orderly repeating forms known as the
a-helix and b-sheet.
The α-helix, as the name implies, is a helical arrangement of a single polypeptide chain
like a coiled spring. In this conformation, the carbonyl and N–H groups are oriented parallel
to the axis. Each carbonyl is linked by a hydrogen bond to the N–H of a residue located four
residues further on in the sequence within the same chain. All C=O and N–H groups are
involved in hydrogen bonds thereby making a fairly rigid cylinder. The α-helix has precise
dimensions of 3.6 residues per turn and 0.54 nm per turn. The side chains project outwards
and contact any solvent, producing a structure something like a bottle brush or a round
hair brush. An example of a protein with many helical structures is keratin that makes up
human hair.
H
H
H
H
H
C
C
C
C
C
C
N
N
N
R
R
R
H
O
O
O
a-Helical structure of protein
The structure of a β-sheet is very different from the structure of an α-helix. In a β-sheet, the
polypeptide chain folds back on itself, so that the polypeptide strands lie side by side and are held
together by hydrogen bonds, forming a very rigid structure. Again, the polypeptide N–H and C=O
groups form hydrogen bonds to stabilize the structure, but unlike the α-helix, these bonds are
formed between neighbouring polypeptide strands. Generally, the primary structure folds back on
itself in either a parallel or antiparallel arrangement, producing a parallel or antiparallel β-sheet.
In this arrangement, side chains project alternately upwards and downwards from the sheet. The
major constituent of silk (silk fi broin) consists mainly of layers of β-sheet stacked on top of each
another.
130
Pharmaceutical Chemistry of Natural Products
HN
RHC
C
NH
CHR
C
N
RHC
C
CHR
N
CH
H
O
H
CH
HCR
N
C
O
RCH
N
C
O
H
HCR
N
HO
C
RCH
N
OH
O
H
H
CH
CHR
N
C
O
RCH
N
H
C
CHR
N
C O
RCH
N
Parallel pleated sheet
O
RCH
C
H
N
CHR
C
O
N
H
O
RHC
O
C
N
H
CHR
C
N
HCR
C
N
H
RCH
C
N
HCR
C
N
H
O
RCH
O
H
H
O
O
H
HCR
C
O
N
RCH
C
N
H
HCR
C
O
N
C
CHR
Antiparallel pleated sheet
Tertiary structure of proteins
This level of structure describes how regions of secondary structure fold together, i.e. the 3D
arrangement of a polypeptide chain, including α-helix, β-sheet and any other loops and folds.
Tertiary structure results from interactions between side chains or between side chains and the
polypeptide backbone, which are often distant in sequence. Every protein has a particular pattern
of folding and it can be quite complex.
Amino acids, Peptides and Proteins
131
Two major molecular shapes have been found in proteins, namely fi brous and globular. Fibrous
proteins have a large helical content and are essentially rigid molecules of rod-like shape. On the
other hand, globular proteins have a polypeptide chain which consists partly of helical sections
and folded about the random coil section to give a spherical shape.
Three bonds responsible for formation of the tertiary structure of a protein are hydrogen,
ionic and hydrophobic. Hydrogen bonds between side chains may arise due to number of residues.
Electrostatic forces may be forces of repulsion or attraction, depending on the charge on the polar
groups. The two polar groups are carboxylate (CO
–
) and ammonium (–NH
3
+
). When like-charged
polar groups are close together, the repulsive forces will destabilize the protein conformation.
On the other hand, when the polar groups have unlike charges, the attractive forces stabilize the
conformation. Such attractive forces are often referred to as ionic bonds or salt linkages.
The hydrophobic bonds are formed between the hydrocarbons like side chains (between two
methyl or phenyl groups) which tend to be in the interior of the protein molecule where there is
less water.
Beta sheet
Disuphide (S-S)
bond
Alpha helix
Quaternary structure of proteins
Majority of the large size and some of the medium-sized proteins exist as an association of several
chains. Here, the protein is known as oligomeric and the individual chains are called protomers
or subunits. Each subunit has its own primary, secondary and tertiary structures, and two or
more subunits in a given protein may have identical or more than three different structures. The
haemoglobin molecule is made up of four subunits (two identical α-chains and two identical
β-chains). The four chains are held together by van der Waals forces (hydrophobic forces) giving
the molecule a quaternary structure that resembles a sphere.