Analysis of Hirudin: Structure, Properties, and Industrial Uses

This report offers a comprehensive analysis of Hirudin, a potent anticoagulant protein. It begins with an examination of the primary structure, identifying key domains and disulfide bond linkages. The report then delves into the physicochemical properties of Hirudin, including its acidic nature, molecular weight, amino acid composition, and hydropathy plot, supported by ExPASy analysis. The historical context of Hirudin's discovery is presented, followed by a detailed description of its purification processes, including ion exchange and gel filtration chromatography, as well as recent advancements using HPLC. The report concludes by exploring the industrial applications of Hirudin, particularly its use in preventing blood clotting, and discusses modern production methods involving recombinant systems and genetic engineering.

Running head: MEDICAL
HIRUDIN
Name of the Student
Name of the University
Author Note

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Table of Contents
Answer 1....................................................................................................................................2
Answer 2....................................................................................................................................3
Answer 3....................................................................................................................................6
Answer 4....................................................................................................................................7
Bibliography...............................................................................................................................9

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Answer 1
The primary structure of Hirudin
N’ terminal end- V-V-Y-T-D-C-T-E-S-G-Q-N-L-C-L-C-E-G-S-N-V-C-G-Q-G-N-K-C-I-L-
G-S-D-G-E-K-N-Q-C-V-T-G-E-G-T-P-K-P-Q-S-H-N-D-G-D-F-E-E-P-E-E-Y-L-Q- C’
terminal end
Important domains and sub-regions
N’ terminal end- V-V-Y-T-D-C-T-E-S-G-Q-N-L-C-L-C-E-G-S-N-V-C-G-Q-G-N-K-C-I-L-
G-S-D-G-E-K-N-Q-C-V-T-G-E-G-T-P-K-P-Q-S-H-N-D-G-D-F-E-E-P-E-E-Y-L-Q- C’
terminal end
In the above structure, there are two domains marked with yellow and red starting from the N
terminal end and the C terminal end respectively.
Domain 1 (Yellow)= Position 1 to 3
Domain 1 is responsible for the interaction of the protein with the active sites of thrombin.
Domain 2 (Red)= Position 55 to 65
Domain 2 is responsible for the interaction with fibrinogen binding exosite of thrombin.
Indication of disulphide connections and glycosylated amino acids
N’ terminal end- V-V-Y-T-D-C-T-E-S-G-Q-N-L-C-L-C-E-G-S-N-V-C-G-Q-G-N-K-C-I-L-
G-S-D-G-E-K-N-Q-C-V-T-G-E-G-T-P-K-P-Q-S-H-N-D-G-D-F-E-E-P-E-E-Y-L-Q- C’
terminal end.
-S-S-
-S-S-
-S-S-

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Disulphide bond linkages-
Positions located in the primary sequence:
1. Position 6 to 14= Red
2. Position 16 to 28= Green
3. Position 22 to 39= Grey
Glycosylation is visible at 45th Threonine residue (O-linked glycosylation).
Answer 2
ExPASy analysis
The mature protein sequence is –
N’ terminal end- V-V-Y-T-D-C-T-E-S-G-Q-N-L-C-L-C-E-G-S-N-V-C-G-Q-G-N-K-C-I-L-
G-S-D-G-E-K-N-Q-C-V-T-G-E-G-T-P-K-P-Q-S-H-N-D-G-D-F-E-E-P-E-E-Y-L-Q- C’
terminal end
Glycoprotein
O linked glycosylation visible at the Threonine residue and thus the protein has O linked or
Oxygen linked glycosylation.
Physico-chemical property of the protein
Hirudin is an acidic protein since it contains more number of negatively charged amino acids
than the positively charged ones. The most significant chemical property of Hirudin is that it
is a 64 amino acid polypeptide which weighs 7000 kDa on an average.
Number of amino acids: 65
Molecular weight: 6969.53
Theoretical pI: 4.04

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Amino acid composition:
Ala (A) 0 0.0%
Arg (R) 0 0.0%
Asn (N) 5 7.7%
Asp (D) 4 6.2%
Cys (C) 6 9.2%
Gln (Q) 5 7.7%
Glu (E) 8 12.3%
Gly (G) 9 13.8%
His (H) 1 1.5%
Ile (I) 2 3.1%
Leu (L) 4 6.2%
Lys (K) 3 4.6%
Met (M) 0 0.0%
Phe (F) 1 1.5%
Pro (P) 3 4.6%
Ser (S) 4 6.2%
Thr (T) 4 6.2%
Trp (W) 0 0.0%
Tyr (Y) 2 3.1%
Val (V) 4 6.2%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0%
(Z) 0 0.0%
(X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 12
Total number of positively charged residues (Arg + Lys): 3
Atomic composition:
Carbon C 287
Hydrogen H 446
Nitrogen N 80
Oxygen O 110
Sulfur S 6
Formula: C287H446N80O110S6
Total number of atoms: 929
Hydropathy plot of Hirudin

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The nature of the predominant group of amino acids in Hirudin is polar and charged amino
acids since most of the cure belongs to the negative quadrant. The hydrophobic amino acids
are very less in number for this protein as compared to the charged amino acids. This is
because of the fact that this protein is an enzyme and can only react with the use of charged
and polar amino acids. Thus, the predominance is found higher in the hydropathy plot given
above. The bulky hydrophobic amino acids include isoleucine, alanine, phenylalanine and
leucine. The charged and polar groups consist of more than 8 types of amino acids which
clearly shows that the overall protein is charged and polar at its active site.
pI value
The overall pI value of the protein is 4.05 which is quite justified since it is mainly composed
of polar and acidic amino acids with also have been plotted in the negative quadrant in the
generated hydropathy plot.

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Answer 3
Discovery of Hirudin
In the past, John Berry Haycraft has been found to be engaged in scientific research which
included blood coagulation as his main factor of the study. In 1884, he was found to have
discovered that leech was responsible for the secretion of a strong anticoagulant. This
anticoagulant was named as Hirudin.
Flow chart of the first purification process
Ion exchange chromatography (with DEAE-C52 colophony)- Process which separates
charged ions and polar molecules according to their affinity with the ion exchangers.
Gel filtration chromatography (with Sephadex-G25 colophony)- This process is used for the
separation of molecules based on their particular size. Molecules larger than a specific size
move out first. Gel filtration chromatography (with Sephadex-G25 colophony)- This process
is used for the separation of molecules based on their particular size. Molecules larger than a
specific size move out first. In the graph stated below, the column value graph on the right
side shows a high level of hirudin activity proving that it has been successfully purified by
column chromatography.
Flow chart of the process has been given after this page.

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Ion exchange
chromatograph
y
Gel filtration
chromatograp
hy
Anti-thrombin activity test
and biuret test (No
explanation since it is a
confirmatory tests, not
purification)
Measurement of
absorption peaks
Buffer solution
was poured
inside the
column.
Ion exchange
resin with
positively
charged ligand
was added
The pure
liquid
supernatant
was collected
from the
eluted solution
and subjected
to further
testing.
DEAE
Sephadex gel
was added to the
column.
Beads of
diameter larger
than the protein
size was
prepared.
Finally eluted
solution was
collected and
subjected to
further testing.

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Answer 4
Use of Hirudin in industries
Hirudin has been discovered long back but has been found to be of great use for the human
population. This compound has both medicinal and therapeutic values due to its nature of the
anticoagulant activity. Anticoagulants function by preventing the coagulation of blood
clotting and will be very much effective in preventing the side effects of drugs such as
heparin. This section will discuss the various uses of Hirudin in the industries. Thrombin has
been found to be one of the most significant blood clotting factors associated with human
blood. Hirudin has been stated to have high specificity and affinity for thrombin. This protein
has been used in industries and investigated in research studies because of its various
therapeutic and medicinal purposes. Various Hirudin analogs and Hirudin has been stated to
have specific anticoagulant activity towards most of the widely used drugs including heparin.
This compound has been used in various industries because of its function in inhibiting
thrombin or Thr that converts fibrinogen to fibrin associated with blood clotting. Thrombin
has been found to be produced by ProThr enzymatic cleavage. Due to the high demand of
Hirudin for the above-stated reason, it has been found that different industries are developing
Hirudin by various recombinant systems including eukaryotes and yeasts. This is the overall
use of Hirudin in the industries.

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Recent production and purification process
HIR gene was inserted into the P. pastoris pPic9K expression vector. The expression of the
gene was under AOX1 or alcohol oxidase promoter control. The coding sequence of HIR
gene was fused to pre-pro alpha mating factor signal sequence. The selection was done by the
presence of Tn903Kanr and HIS4+ genes in pPic9K.The recombinants were recovered by
HIS+ gene presence and plated in a medium with increased concentrations of
aminoglycosides.The resulting transformants including His+ G418 resistance have been
found to grow in shake flasks and then screened for secretion of recombinant Hirudin. Clones
showing the production of HIR has been found to secrete 1.5 g/L Hirudin.Chromatography
was used in a similar way as the older methods for 98% purification and 63% recovery
percentage. HPLC- High-performance liquid chromatography has been used as a technique
for the separation, identification and purification of the specific components of a mixture.
This process has been found to rely on a pump which forces the pressurized liquid to pass
through the column filled with an adsorbent. When the liquid component interacts with the
solid adsorbent material, flow rates become different and the components are well separated
on the basis of their elution speed from the column. In the figure given on the left-hand side,
it has been observed that there are three different purification absorbance curves. The first
curve shows the purification peaks of the filtered broth which consist of a large number of
impurities. The second one shows the separation by gel filtration chromatography which also
shows less purification. However, the last one shows a high level of purification with a single
peak of HPLC due to the efficiency of this chromatographic procedure. In this way,
recombinant Hirudin is generated and purified in today's industries.
Flow chart of the process

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