Protein is a macronutrient that is essential to building muscle mass. It is commonly found in animal products, though is also present in other sources, such as nuts and legumes. There are three macronutrients: protein, fats and carbohydrates. Macronutrients provide calories, or energy.

1. Protein 2
Md. Saiful Islam
B.Pharm, M.Pharm (PCP)
North South University
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2. Determination of Amino acid sequence in a polypeptide chain:
There are six basic steps to determine the amino acid sequence of a
polypeptide chain.
Step 1: Deermining the amino acid composition.
The first step is to hydrolyze all the peptide bonds of the pure
polypeptide chain. Hydrolysis is done by heating a polypeptide chain
in 6M HCl at 100-110°C for 24 hours or longer. Proteins with many
bulky hydrophobic groups may require longer heating periods. The
amino acid mixture so formed can be separated by ion exchange
chromatography to determine which amino acids are present and
the relative numbers of each amino acids.

3. Step2: Identifying Amino- and Carboxy-Terminal
amino acids:
To identify the amino-terminal amino acids Sanger developed the
reagent 1-fluoro-2,4-dinitrobenzene which can lebel the amino-
terminal residue of the chain as the yellow 2,4-dinitrophenyl (DNP)
derivative. When such a DNP derivative of a polypeptide is subjected
to hydrolysis with acid, all the peptide bonds in the chain are
hydrolyzed. However, the covalent bond between the 2,4-
dinitrophenyl group and the -amino group of the amino-terminal
residue is resistant to this treatment. Consequently, the amino-
terminal residue will be present in the hydrolysates as its 2,4-
dinitrophenyl derivative. This derivative can be separated easily
from the unsubstituted free amino acids and identified by
chromatographic comparison with authentic dinitrophenyl derivatives
of the different amino acids.

4. Sanger's method of N-Terminal amino group analysis:
A: derivatization of N-terminal end with Sanger’s (DNFB),
B: Total acid hydrolysis of the dinitrophenyl peptide

5. Another reagent used to label the amino-terminal residue is dansyl
chloride which reacts with free -amino group to yield a dansyl
derivative. This is highly fluorescent and can thus be detected and
measured in much lower concentration than dinitrophenyl derivatives.
The carboxy-terminal amino acid residue of a polypeptide
can be identified through incubation of the polypeptide chain with
the enzyme carboxypeptidase, which hydrolyzes only the peptide
bond at the Carboxy-terminal end of the chain. By determining
which amino acid is released first by the action of carboxypeptidase
on the polypeptide, the carboxy-terminal residue can be identified.

6. Step 3: Fragmenting the Polypeptide Chain
Another sample of the intact polypeptide chain is now
fragmented into smaller pieces, short peptides having on an
average of 10 to 15 amino acid residues. The objective is to
separate these fragments and determine the amino acid sequence
of each.
A common procedure is partial enzymetic hydrolysis of the
polypeptide by the digestive enzyme trypsin which catalyzes the
hydrolysis of only those peptide bonds in which the carboxyl
group is contributed by either a lysine or an arginine residue.
Now from these fragments total number of lysine or arginine can
be predicted. The fragments produced by the action of trypsin
can be separated from each other by ion exchange
chromatography or by paper electrophoresis.

7. Step 4: Identifying the sequences of the peptide fragments
The amino acid sequence of each peptide fragment resulting from
step 3 can be determined by the Edman degradation procedure which
labels and removed only the amino terminal residue from the peptides,
while leaving all the other peptide bonds intact.
In Edman degradation procedure the peptide is first reacted with
phenylisothiocyanate which combine with the free -amino group of
the amino-terminal residue. Treatment of the peptide with cold dilute
acid (HCl) removes the amino-terminal residue which can now be
identified by chromatograpy. The rest of peptide chain remains intact.
The shortened peptide can now be subjected to another round of
these reactions, which permits the identification of the new amino-
terminal residue. In this method the amino acid sequence of the
petides can be identified.

8. Step 5: Cleavage of the original polypeptide chain by a
second procedure
In order to establish the order of the peptide fragments, formed by
trypsin, another sample of the intact polypeptide is cleaved into small
fragments by a different method. The reagent cyanogen bromide is
particularly useful, since it cleaves only those peptide bonds in which
the carboxyl group is contributed by methionine residues. Thus if the
polypeptide contains eight methionine residues, cleavage with cyanogen
bromide will usually yield nine peptide fragments. The fragments
resulting from this procedure can now be separated by each other using
electrophoresis or chromatography. Each of these small fragments are
now subjected to the repeatitive Edman degradation as in step 4 to
identify its amino acid sequence.

9. Step 6: Ordering peptide fragments by establishing
overlaps
The sequences of amino acids in each fragment obtained from the
original polypeptides by the two cleavage procedures are examined to
find out the continuation or overlapping. Overlapping peptides obtained
from the second fragmentation yield the correct order of the peptide
fragments produced in the first cleavage. In this process the whole
sequence of the polypeptide chain can be determined.
Sometimes third or even fourth cleavage method may be needed to
obtain the complete sequence.
Enzyme Cleavage points
Trypsin Lysine, Arginine
Chymotrypsin Phenylalanine, Tryptophan, Tyrosine
Cyanogen bromide Methionine

10. Glucogenic and ketogenic amino acids:
A. Glucogenic amino acids: Amino acids whose catabolism yields
pyruvate or one of the intermediates of the citric acid cycle are
termed glucogenic or glycogenic. These intermediates are
substrates for gluconeogenesis in the liver or in the muscle.
B. Ketogenic amino acids: Amino acids whose catabolism yields either
acetoacetate or one of its precursors (acetyl Co-A or acetoacetyl
Co-A) are termed ketogenic amino acids. Acetoacetate is one of the
keton bodies which also includes 3 hydroxy butyrate and aceton.
Leucine and lysine are the only exclusively ketogenic amino acids in
the protein.
Glucogenic Ketogenic
Nonessential Ala, Arg, Asn, Asp, Cys, Glu, Gln
Gly, Pro, ser, Tyr*
Essential His, Met, Thr, Val, Ileu*, Phe*, Trp* Leu, Lys
*both glucogenic and ketogenic

11. Pyruvate
Ala, Thr, Gly,
Ser, Cys
Phe, Tyr, Leu,
Lys, Trp
Acetoacetyl
CoA
Asp, Asn
Phe, Tyr
Ileu, Met,
Val
Glutamate
Fate of Amino acids /
Degradation of Carbon
skeleton of amino acids:
Arg, His, Gln, Pro

12. Urea cycle
The urea cycle is a cycle of biochemical reactions occurring in many
animals that produces urea (H2N-CO-NH2) from ammonia (NH3). One
nitrogen of the urea molecule is supplied by free NH3 and the other
nitrogen by aspartate. The carbon and oxygen of urea are derived from
CO2. Urea is the major disposal form of amino groups derived from
amino acids, and accounts for about 90% of the nitrogen containing
compounds in the urine. This cycle was the first metabolic cycle
discovered (Hans Krebs 1932). In mammals, the urea cycle takes place
primarily in the liver and then is transported in the blood to the
kidneys for excretion in the urine.
Organisms that cannot easily and quickly remove ammonia usually have
to convert it to some other substance, like urea which are much less
toxic.
Overall reaction of the Urea Cycle:
Asp + NH3 + CO2 + 3ATP Urea + fumarate + 2ADP + 2Pi +AMP+ PPi +
3H2O

14. Nitrogen Balance:
Most of the N2 in the diet is consumed in the form of protein.
Nitrogen balance occurs when the amount of nitrogen consumed
equals that of nitrogen excreted in the urine, sweat and feces. Most
healthy adults are normally in nitrogen balance.
Positive Nitrogen balance: This occurs when nitrogen intake exceeds
nitrogen excretion, It is observed during situations in which tissue
growth occurs, for example, in childhood, pregnancy or during
recovery from an illness.
Negative Nitrogen balance: This occurs when nitrogen loss is greater
than nitrogen intake. It is associated with inadequate dietary
protein, lack of essential amino acid or during physiologic stresses
such as trauma, burns, illness or surgery.

15. Amino acids in disease diagnosis:
There are a number of clinical disorders in which a high concentration of
certain amino acids are found in the plasma and urine. An abnormally high
concentration of amino acids in urine is called an aminoaciduria.
Phenylketonuria: Defects in Phe metabolism which results high
concentrations of Phe, phenylpyruvate and phenyl lactate accumulate in
the plasma and urine. Phenylketonuria occurs clinically in the first weeks
after birth and if the infant is not placed on a special diet, severe mental
retardation will occur.
Cystinuria: Defects in metabolism of cystine and basic amino acids (Lys,
Arg, Ornithine) and large amounts of these amino acids are excreted in
the urine. Other symptoms may arise from formation of kidney stone.
Maple syrup urine disease: Defects in the metabolism of Leu, Ileu and
Val, and therefore accumulates in the blood as large amounts, causing a
toxic effect that interfere with brain function. If untreated the disease
leads to mental retardation, physical disabilities and even death. Patients
are experienced to characteristic maple syrup odor to the urine.