Thesis submitted to the partial fulfilment of the requirements of the award

of the degree of MASTERS OF SCIENCE IN BIOTECHNOLOGY to

the

Dr. Babasaheb Ambedkar Marathwada University Aurangabad

By
Sunoj suresh
Aishwarya Udawant
Abhimanyu Katkar
 Certificate

This is to certify that this dissertation entitled on submitted

to Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad, in partial fulfillment of the requirement for the
award of the degree of Bachelors of Science in Biotechnology
embodies the results of bonafide project work carried out
by Sunoj Suresh, Aishwarya Udawant and Abhimanyu
Katkar under the guidance of Mr. Faiyaz Shaikh, Assistant
Professor, MGM's Institute of Biosciences and
Technology,Aurangabad. It is further certified that no part
of the dissertation has been submitted anywhere else for any
degree, diploma and associate of fellowship.

Dr. Sanjay Harke Mr. Faiyaz Shaikh

EXAMINER
DIRECTOR PROJECT GUIDE
 Certificate

This is to certify that this dissertation entitled on

submitted to Dr. Babasaheb Ambedkar Marathwada
University, Aurangabad, in partial fulfillment of the
requirement for the award of the degree of Masters of
Science in Biotechnology embodies the results of bonafide
project work carried out under the guidance of Mr. Faiyaz
Shaikh, Assistant Professor, MGM's Institute of Biosciences
and Technology, Aurangabad. It is further certified that no part
of the dissertation has been submitted anywhere else for
any degree, diploma and associate of fellowship
Sunoj Suresh Aishwarya Udawant Abhimanyu
Katkar
Acknowledgement
Foremost, I would like to express my sincere gratitude towards
our director, Dr. Sanjay Harke for granting us the opportunity
to work in the laboratories of this Institution
Next, I would like to express my gratitude towards Mr. Faiyaz
Shaikh, our project guide, for his continuous support towards
my project, for his patience, motivation, enthusiasm, and
immense knowledge. His guidance helped me in all the time
of project work and writing of this thesis. Besides my director
and my project guide, I would like to give a special thanks
to the teaching and the non-teaching staff including Mr
Dadarao Lokhande, Ms Trupti Kadam, Mr Arjun, Mr. Krishna
Chavan, for providing me with all the necessary chemicals I
needed for my project work. I would also like to thank my
project members who have been a constant support all
through the time and worked together as a team and for the fun
time we spent together doing our project.
Last but not the least, I would like to thank my parents and
friends who have been a source of motivation and enthusiasm
and without whom I would have never been able to reach these
heights.
.
 Sunoj Suresh
Aishwarya
Udawant
Abhimanyu
Katkar

LIST OF ABBREVIATIONS:

G Gram

KDa Kilodalton

M Molar

PI Protease Inhibitor

GXCP Gel X ray contact Print Technique.

g microgram

l Microlitre

ml millilitre

mi Minute

OD Optical density

PAGE Polyacrylamide Gel Electrophoresis

Tris HCl Tris aminomethane- Hydroxy chloride

 CONTENTS

CHAPTER PAGE NO.

TITLE
NO.
I. INTRODUCTION
II. REVIEW OF LITERATURE
III. MATERIALS AND METHODS
IV. RESULTS
V. DISCUSSION
VI. SUMMARY
REFERENCES
APPENDIX
 LIST OF FIGURES

FIGURE PAGE NO.

TITLE
NO.
 LIST OF TABLES

PAGE NO.
TABLE NO. TITLE
 LIST OF PLATES

PAGE NO.
PLATE NO. TITLE
 Introduction

Proteases (EC 3.4) are one of the principal families of enzymes which
selectively catalyse the hydrolysis of peptide bonds in proteins (Fan and Wu, 2005).
Proteases diversely occur in every living creature and found to adapt wide range of
conditions (variations in pH, reductive environment and so on). Proteases participate
in most aspects of cell nutrition, physiology, signalling cascades, cell migration, cell
invasion and microbial pathogenesis (Ward, 2009). Their contribution in food
digestion, blood clotting, embryogenesis, tissue reorganization (e.g. wound healing,
regeneration, molting, metamorphosis etc.), defense mechanisms and immune
responses are well established (Ivanovo et al., 2006; Ryan, 1990). It is estimated that,
around 1-2% genes on genome of various organism code for proteases (Barrett et al.,
2001).
Protease action can be divided into two categories: limited proteolysis and
unlimited proteolysis. In limited proteolysis, proteases chop off definite peptide
bonds in adolescent proteins or remove the target signals in proproteins. In unlimited
proteolysis, proteins are chopping off into their amino acid constituents. Limited
proteolysis is obligatory for fine control of protein metabolism like post-translational
modification. Unlimited proteolysis leads to bulk hydrolysis of proteins which
provide simple metabolites (amino acids) to cell, essential for growth and
development. If proteolytic control is lost, proteases would be destructive to the cell
or organisms hence proteolytic activities precisely regulated in the cell (Fan and Wu,
2005).
Proteases can be grouped into endopeptidases (EC 3.4.21-24) and
exopeptidases (EC 3.4.11-19). Endopeptidases cleave the internal peptide bonds and
exopeptidases cleave only the N-terminal or C-terminal bonds of proteins.
On the basis of mechanism of catalysis and the amino acid present in the active
centre, endopeptidases can be classified into serine proteases, aspartic proteases,
cysteine proteases and metalloproteases (Barrett et al., 2001).
Among all endopeptidases, serine proteases are widespread in insects,
mammals, viruses, bacteria and animals. They are involved in tissue degradation,
blood coagulation, digestion, development and immune defense (Molehin et al.,
2012). Serine proteases (trypsin, chymotrypsin and elastase) are abundantly present in
the alkaline gut of various Lepidopteron larvae where they are playing important role
in proteinaceous food digestion (Terra et al., 1996). Almost all phytophagus insect
pests utilize this proteolytic machinery for feeding on agriculturally important crops.
With plasticity in expression of serine proteases, insect pests easily adapts on various
plants to acquire nutrition (Patankar et al., 2001).
Protease inhibitors (PIs) are molecules that resist the proteolytic actions of
proteases and play a vital role in regulation of protein catabolism. They have been
studied by different researchers around the world because of its ability to stop
proteases from being destructive (Give REF). They are small proteins which are quite
common in nature and have molecular weights ranging from 4 to 85 kda (Hung et. al.,
2003; Fritz, 2000). PIs bind at the active site of the proteases, resulting in the
formation of a stable tetrahedral intermediates protease-inhibitor complex and loss of
enzymatic activity (Bode and Huber, 2000).
Protease inhibitors are widely dispersed in plant tissues, abundantly in seeds
(about 10%) (De Leo et. al., 2002). A detectable amount of PIs expression is also
reported from flowers and leaves (Padul et al., 2012). The upregulated activities of PIs
are also seen by insect pests or herbivore attacks. The physiological functions of PIs
in plants include, regulation of intracellular protein homeostasis, acting as storage
proteins and plant protection agents against insect pests and pathogen attack (Ryan,
1990). PIs considered as plant defensive compounds, as they slow down the growth of
many Lepidoptera insect pests by arresting the activity gut proteases (Broadway,
1995).
Besides their significant role in plant, PIs have different applications in
medicines, many proteases are essential for propagation of diseases, and hence
inhibition of proteases is emerging as a promising approach in medicinal application
for cancer, obesity, hepatitis, herpes, cardiovascular, inflammatory, neurodegenerative
diseases, and various infectious and parasitic diseases. Irrespective of their key roles,
they need to be checked regularly by PIs as their excess expressions may otherwise
alter the normal functioning of cells.
 The applications of PIs have been vast, which is currently on the
verge of becoming one of the most studied areas in the research world.
Protease inhibitors have different applications in medicines, plant pest
control, agriculture, treatment of HIV etc. Protease inhibitors have played
a pivotal role in controlling the different proteases which can otherwise
alter the normal functioning of cells.
Many proteases are also essential for propagation of diseases, and hence
inhibition of proteases is emerging as a promising approach in medicinal
application for cancer, obesity, hepatitis, herpes, cardiovascular,
inflammatory, Neurodegenerative diseases, and various infectious and
parasitic diseases (Rao et al. 1998).
Plants have elaborated protective mechanisms that allow them to
successfully resist different kinds of unfavourable conditions including
insects and phytopathogenic microorganisms. The most important
components of all protective mechanisms are based on proteinaceous
compounds. Protease inhibitors (PIs) are widely dispersed in plant
tissues, often occurring in quite high concentrations (Murdock and Shade
2005). They are an important element of the plant defense response to
insect predation, and may also act to restrict infection by some
nematodes.
Recent achievements in biotechnology resulted in creation of transgenic
plants with an increased resistance towards different kinds of
unfavourable conditions including effects of phytopathogenic
microorganisms and viruses.

Protease Inhibitors are plant defensive proteins that.

Plant proteinase inhibitors are also thought to help to arrest pathogen

invasions by inhibiting proteolysis thereby limiting availability of amino
acids for growth and multiplication of the pathogen (Valueva and
Mosolov, 2004).
HOW PROTEINASE INHIBITORS AFFECTS DIGESTION:

The key role of proteinases in the digestive processes of animals and

microbes have generated considerable interest about the effects of
proteinase inhibitors that are often present in the food chain of human
(Ryan, 1990 ). After it became established that plants produce PIs in
response to attack by herbivores, researchers investigated the effects of
these inhibitors on fitness parameters of herbivores.

Protease inhibitors are divided in following families

1. Serine PIs
2. Cysteine PIs
3. Aspartic PIs
4. Metallo PIs (Laskowski and Kato, 1980)

Serine Proteinases Inhibitors:

Serine Proteinase inhibitors were firstly discovered in reserve organs of
plants which are utilised by man and animals as a source of protein, for
this reason seeds and tubers were early sources of new inhibitors of plant
origin. Inhibitors were lately found in other organs like leaves and fruits. (
Xavier- Filho and Campos 1989).

Serine proteinase inhibitors associate with their related enzymes (

Laskowski and Kato 1980).
The enzymes react with the inhibitor molecule as it were a proper
substrate and remain bound in a strong covalent complex through reactive
site .

Among all these PIs serine Protease Inhibitors are Predominantly found
in all plants.

Serine Protease inhibitors have been classified into 9 sub families.

The 9 sub families are as follows:

1. Kunitz
2. Bowman- Birk
3. Cucurbitaceae
4. Potato 1 (PPI-1)
5. Potato 2 (PPI-2)
6. Superfamily of cereal Inhibitors.
7. Mustard Trypsin Inhibitor
8. Serpin
9. Amylase Inhibitor of Alpha amylase and trypsin of cereals.

Kunitz and Bowman-Birk Inhibitors

Kunitz and Bowman Birk Inhibitors are most commonly found in

members of Leguminosae Family. These Inhibitors are distinguished on
the basis of their cysteine content and number of reactive sites.

Kunitz Inhibitors (18-24 kda) either have one or two polypeptide chain( s
) with four cysteine residues forming two disulphide bridges and have
single reactive site, on the other hand Bowman-birk Inhibitors are
relatively small proteins (4-8 kda) with 14 cysteine residue forms 7
intramolecular Di- sulphide bridges. (Vinayak R. Tripathi et.al. 2013)

Inhibitors from cucurbitaceae Family

Seeds of plants of family Cucurbitaceae like the squash, Cucumber and

zucchini contain inhibitors of trypsin. ( Richardson 1991)

Inhibitors from potato Tubers.

The potato (Solanam tuberosum) tuber is very rich source of serine
Proteinase Inhibitors.( Ryan 1981 and Richardson 1991). Potato Inhibitor
1 is very small (8kda).

1) Potato type 1 PIs- Inhibitors of these families are widespread in

plants including potato tubers (Ryan and balls 1962) Tomato fruit
(Margossian et al. 1988).

These inhibitors have molecular mass of 8 kDa.

These inhibitors are monomeric.

Inhibitors in this family lack any disulphide bridges (Cai et al.

1995)

Inhibitory mechanism in these families is considered to fit the

standard
model.

2) Potato type 2 PIs- The members of this group are from solanaceae
family.
These inhibitors found in leaves, flowers, fruits and phloem of
other solanaceae species.(Iwasaki et al. 1971)
An analysis of these inhibitors and genes has shown that they are
composed of multiple repeat units ( Antcheva et al. 2001; Miller et
al. 2000)
Inhibitors in this family have been reported to inhibit chymotrypsin
trypsin, Oryzin (Antcheva 1996)

Homologues of these inhibitors are found in leaves of solanaceae

family,
in seeds of barley ( Richardson 1991)

Inhibitors from cereal seeds:

Seeds of cereals (Gramineae ) are rich source of serine proteinase

Inhibitor of most of several inhibitor families. Some of these cereal
 inhibitors are bi-functional i.e. they inhibit serine proteinases as well
as inhibit alpha amylases.( campos and Richardson 1983).

Mustard trypsin inhibitor (MSI )

These are small single polypeptide chain inhibitors with the molecular
mass about 7 kDa .

These inhibitors found in family Cruciferae (Laing and McManus 2002)

These inhibitors have been isolated and characterised from a number of

species including white mustard (Sinapis alba) and Tape ( Brassica
napus)(Ascenzi et al. 1999)

These inhibitors forms tight binding complex with trypsin and apparently
follow standard mechanism( Ceciliani et al. 1994)

Common Type of Example Source Target protease References

name
Kunitz Soyabean Kunitz trypsin Glycine max Trypsin, Laskowski and
inhibitor Hordeum chymotrypsin kato(1980)
Barley subtilisin inhibitor vulgare Subtilisin,Alpha- Vallee et
Winged-bean chymotrypsin Psophocarpus amylase al.(1998)
inhibitor Tetragonolobus Alpha- Habu et
Kunitz cystein peptidase Solanum chymotrypsin al.(1982)
inhibitor 1 tubersum Cysteine protease Gruden et
al.(1997)
Cereal Ragi seed trypsin/a-amylase Eleusine Alpha-amylase Hojima et
 inhibitor coracana Alpha- al.(1980)
Barley trypsin/factor Xlla Hordeum vugare amylase,trypsin Lazaro et
inhibitor Triticun Alpha-amylase, al.(1988)
Wheat trypsin/alpha- aestivum trypsin Shewry et
amylase inhibitor Zea mays Mammalian al.(1984)
Maize trypsin/factor Xlla trypsin, activated Mahoney et
inhibitor hageman factor al.(1984)
Potato Chymotrypsin inhibitor l Solanum Chymotrypsin, Richardson
type 1 Glutamyl peptidase ll tuberosum trypsin Glu (1974)
inhibitor Momordica S.griseus protease, Ogata et al.
Subtilisin-chymotrypsin charantia subtilisin (1991)
inhibitor Cl-1A Hordeum vugare Subilisin, Greagg et al.
Wheat Triticum chymotrypsin (1994)
Subtilisin/chymotrypsin aestivum B.lichenoformis Poerio et al.
inhibitor subtilisin, Alpha- (2003)
chymotrypsin
Mustard Mustard trypsin inhibitor Sinapis alba Beta-trypsin Menegatti et al.
Mustard trypsin inhibitor-2 Brassica hirta Bovine beta- (1992)
Rape trypsin inhibitor Brassica napus trypsin,Alpha- Ceci et al.
chymotrypsin (1995)
Trypsim, Ceciliani et al.
chymotrypsin (1994)

Cystatin Onchocystatin Onchocerca Cysteine proteinase Lustigman et al.

Ovocystatin vovulus Thiol proteases (1992)
Oryzacystatin ll Gallus gallus Cysteine Laber et al.
Oryza sativa proteinases (1989)
Ohtsubo et al.
(2005)
Bowman- Bowman-birk plant trypsin Glycine max Trypsin, Odani and
Birk inhibitor unit 1 Arachis chymotrypsin Ikenaka (1976)
Bowman-birk hypogaea Trypsin, Suzuki et al.
Trypsin-chymotrypsin Helianthus chymotrypsin (1987)
inhibitor annuus Trypsin, cathespin Mulvenna et al.
Sunflower cyclic trypsin G, (2005)
inhibitor Elastase,
chymotrypsin and
thrombin
Potato Proteinase inhibitor ll Solanum Trypsin, Greenblatt et
type 2 Potato peptidase inhibitor ll tuberosum chymotrypsin al. (1989)
inhibitor unit 1 Solanum Trypsin, Keil et al.
Tomato peptidase inhibitor tuberosum chymotrypsin (1986)
ll inhibitor unit 1 Solanum Trypsin, Graham et al.
Tomato peptidase inhibitor lycopersicum chymotrypsin (1985)
ll inhibitor unit 2 Trypsin, Barrette-Ng et
chymotrypsin al. (2003)
 Cysteine proteinase Inhibitor: (The Cystatin
superfamily)

The cystatin superfamily composed of several families and includes

proteins that are related in structure and function to an inhibitor of
cysteine proteinase.

The members of these families inhibit the activity of cysteine protease

and called cysteine PIs or cystatin.

They are widely distributed in plants. (Oliveira 2003).

These inhibitor are grouped into four families based on molecular mass
and disulphide bonds numbers and arrangement. (Turk and Bode 1991)

1) Family 1 Cystatins (Stefin)

2) Family 2 Cystatins (Cystatin family)
3) Family 3 Cystatins ( Kininogen family)
4) Family 4 Cystatins (Phytocystatins)

Cysteine proteinases apparently more important than serine proteinases in

the mobilization of proteins during germin ation of seeds and other
processes ( Baumgartner and chrispeels, 1976; shutov and Vaintraub,
1987; Huffaker, 1990).

3)Aspartic proteinase inhibitor-

Knowledge on the role of aspartic proteinases in insect digestion is

limited than that of cysteine proteinases. Aspartic proteinases arent
active at pH 8-11 (Houseman et al. 1987)

Potato tubers possess an aspartic proteinase inhibitor.

Low pH of midgut of many members of coleopteran and hemiptera

provides more favourable envioronment for aspartic proteinases.

4)Metallo-proteinases inhibitor
Two families of Metallo-proteinase inhibitor

1) Metallo- carboxypeptidase inhibitor family in potato and

tomato plant.
2) Cathepsin- D inhibitor family in potatoes inhibits trypsin and
chymotrypsin and cathepsin-D. It inhibits broad spectrum of
carboxypeptidase.

Inhibitors of the Metallo carboxypeptidase from tissue of

tomato and potato are polypeptide that strongly and
competitively inhibits broad spectrum of carboxypeptidase.

Cathepsin- D inhibitor-
Its of 27 kDa.
It inhibits trypsin and chymotrypsin as well as Cathespin D.
The inhibitor is found in tissue of potato tubers where it
accumulates during tuber development along with potato
inhibitor 1 and 2 families of serine proteinase inhibitor.
The inhibitor also accumulates in potato leaf tissue.
Inhibitor Pest Reference
Bowman-birk Teleogrylleus Burgells et al. 1991
inhibitor commodus
Kunitz Helicoverpa Ishikawa et al. 1994.
armigera Spodoptera McManus and
litura Burgess 1995
Johnston et al. 1993
Potato Inhibitor 2 Chrysodeisus McManus et al.
Erisoma 1994 Burgess et al.
T. commodus 1999

Table 1: Pesticidal activity of plant protease inhibitor

 Mechanism of Toxicity of protease inhibitors
Mechanism of these proteinse inhibitor has been subject of
intense investigation.

In insects injested food proteins triggers the synthesis of and

release of enzyme from posterior to midgut epithelial cells.
The enzymes released from membrane associated forms and
sequestered in vesicles that are in turn associated with
cytoskeleton.

The peptidase secreated into ectoperitopic space between the

epithelium as a particulate complex from where proteases move
transversely into lumen of gut where the food is degraded.

PI inhibits the protease activity of enzyme and reduces quantity

protein that can be digested as result insects become weak with
stunted growth and ultimately die.

Protease inhibitor assay

Activity of protease inhibitor against protease was assayed. In
this method, the TCA soluble fractions formed by action of
trypsin on the protein substrate Hammerstein casein was
measured by the change in Absorbance at 280nm. The residual
caseinolytic activity of the trypsin in the presence of inhibitor,
at 37C,was used as a measure of inhibitory activity,
Appropriate blanks for enzyme, inhibitor, and substrate were
also included in the assay along with the test.
 Mechanism of binding of plant protease inhibitor to insect
proteases appear to be similar with all four classes of inhibitor
binds to active site on the enzyme and forms complex with low
dissociation constant.
Effective blocking of inhibitors thus mimic normal substrate for
enzyme but doesnt allow the normal enzyme mechanism of
peptide bond cleavage to proceed completion i.e. dissociation of
product.

Reagents for PI Assay:

1) Trypsin 1mg/ml
50mg trypsin was added in 50ml of D/w.
2) Casein 1%
1gm casein was dissolved in 100ml D/W with gentle Warm
heating.
3) Tris buffer 1M
4) TCA 10%
5gm TCA dissolved in 50ml D/W

MATERIALS AND METHODOLOGY

 1) Procurement of sample seeds and chemicals-

Dry seed sample were collected from Pandariba, Aurangabad 431001

(MS), India. Ammonium persulfate, hexane, acrylamide, bisacrylamide,
Tetramethylethylenediamine (TEMED), polyvinylpyrolidone (PVP) were
obtained from MGMIBT laboratory Aurangabad, India. Gelatin coated X-
ray film were provided by Mr. Faiyaz Shaikh, Assistant professor,
MGMIBT.

2) Extraction of crude proteases:

-Mature, dried seeds of above samples were pulverised to a fine powder

by a mixer grinder.

-The Fat was removed from the powder by hexane and acetone washes.
Sample powder and Hexane were taken in 1:10 proportion and incubated
overnight. This procedure removes fat.

-sample powders were suspended in chilled acetone for three hours.

-Defatted powder was suspended In milli-Qwater (1:15 w/v) containing

1% PVP and incubated overnight at 150C.

-The suspension was then centrifuged at 12000 g for 20mins at 40C and
supernatant was termed as crude protease inhibitor

Activity of Trypsin :
 [Image 1]
Activity of trypsin at 9 min.

[Image 2]
Activity of trypsin 8 min.

[Image 3]
Activity of trypsin 7 min.

Activity of trypsin( 1mg/ml ) was checked at different time

period. The ideal activity of Trypsin was found at 7 mins.
 Dot Blot Assay or spot assay:

Analysis of protease inhibitor by Dot Blot method

The purified fraction collected from ion exchange
chromatography,
was analyzed for its protease inhibitory activity according to the
method of
Veerappa et al., (2002) as described below.
I. 3~.d ofprotease inhibitor was mixed with 3:1,1:3,1:1 trypsin
(1mg/ml).
And spotted on to a strip of X-ray film.as the control and
spotted on to the X-ray film.

Ill. Incubated the X-ray film at 37C for 7 minutes.

IV. Washed the film under tap water till the zone of gelatin
hydrolysis by trypsin was visualized.

V. Where the inhibitor is present, the trypsin does not degrade

the gelatin on the x-ray film.If the inhibitor is absent, a clear
zone is formed at the site of sample application on the X-ray
film.

Dot blot test was carried out to determine the potency of

protease inhibitor against trypsin using x-ray film (Pichare and
Kachole 1994; Padul et al. 2012)

The total volume was, made up to 20l by using respective

buffer and loaded onto x-ray film.
The film with spots was incubated for 8min at 370C, then
washed with tap water and dried in air.

Different ratios of enzyme and inihibitor produced different

patterns of gelatin hydrolysis on the X- ray film depending on
effiency of inhibitors.
Figure 1: Dot blot assay of trypsin interactions with PI extract of.
Different ratios of trypsin and trypsin inhibitor were incubated and loaded
on X-ray film.

(B)
 (C)

Protein estimation by folin- lowry method:

Lowry protein assay is biochemical assay for determining the total level
of protein in a solution.

The total protein concentration is exhibited by a colour change of sample

solution in proportion to protein concentration, which can then be
measured using colorimetric technique.

It is named for the biochemist Oliver H. Lowry who developed reagent in

1940s.

Mechanism-
Method combines the reaction of copper ions with the peptide bonds
under alkaline conditions. Divalent copper ions reduced to
monovalent ions.
Monovalent copper ions under radical group of aromatic amino
acids like tyrosine, tryptophan and cysteine react with folin reagent
to produce an unstable product that reduced to Tungsten blue.

Reagents-
 [A] Lowrys reagent
(a) 0.1 N NaOH 0.4gm in 100ml D/W
(b) Na2Co3 2gm in 100ml 0.1N
NaOH
1gm Na2CO3 was added in 0.1N NaOH

[B]1% CuSO4 0.5gm CuSO4 in 50ml

[C] 2% Sodium potassium tartarate1gm Sodium potassium

tartarate in 50ml D/W.

[D]0.5ml of reagent B and 1 ml of reagent C were mixed

together and 50ml of volume was made by reagent A. This
forms reagent D.

[E] Protein stock- 100mg of BSA was added in 20ml of D/W.

From this 5ml was added in 95 ml of D/W . This forms standard
of 25mg/ml.

D/W Lowry Folin O.D. @

ml of Std (ml) reagent Phenol 660nm
(ml) (ml) Reagent
(ml)
0 1 0
0.2 0.8 0.15
0.4 0.6 20min 0.30
0.6 0.4 10 0.5ml 0.46
0.8 0.2 0.61
1.0 0 Min 0.70
Sample1 0.9 0.67
(0.1) 3ml
Sample2 0.9 0.35
(0.1)
Sample3 0.9 0.78
(0.1)
 Table 2 : Folin-lowry reaction

CHARACTERIZATION OF PROTEASE INHIBITOR

Purified inhibitor was further subjected to characterization for their

biophysical and physicochemical properties like molecular weight.

NATIVE PAGE (Polyacrylamide gel electrophoresis)

Principle:

Cross linked polyacrylamide gel are formed from the polymerization of

acrylamide monomer in the presence of smaller amount of N,N
methylene disacrylamide (Normally referred to as bisacrymide) used as
cross-linking agent.

Acrylamide monomer is polymerised in head to tail fashion into the

growing chain and occasionally bisacrylamide molecule built into
growing chain, thus introducing second site for chain extension.

The polymerization of acrylamide is an example of free radical catalyst

and is initiated by addition of ammonium persulphate and the base
N,N,NN Tetramethylenediamine (TEMED)

TEMED catalyses the decomposition of the persulphate ion to give a free

radical that is a molecule with a unpaired electrons.

Free radicals are highly reactive species due to presence of an unpaired

electron that needs to be paired with another electron to stabilize the
molecule.

Long chains of acrylamide are built up, being cross linked by introduction
of occasional bisacrylamide molecule into growing chain.

The pore size in gel can be varied by changing the concentrations of both
acrylamide and bisacrylamide.

Acrylamide gels can be made with a concentration between 3% to 30%

acrylamide. Thus low percentage gel eg 4% have large pore size and used
for electrophoresis of proteins.
Low percentage of acrylamide gels are also used to separate DNA. Gels
between 10%-20% acrylamide are used in techniques like SDS Gel
electrophoresis.

Electrophoretic methods

The crude buffer extract of protease inhibitor prepared from dried

seeds,protease inhibitor obtained.

Reagents for polyacrylamide gel electrophoresis :

Reagents for polyacrylamide gel electrophoresis

1) Stock acrylamide solution: ( 30%)

Acrylamide _ 75 gm

Bis-acrylarnide _ 2.6 gm

Distilledwater (DW) _ 250.0ml

Stored at 4C in amber coloured bottle

2) Stacking gel buffer stock:

Tris buffer (1 M) _ 30.3 g

pH (6.8)

0.1 N Hcl

DW _ 250 ml

3) Resolving gel buffer stock:

Trisbuffer (1.5 M) _ 30.3 g

0.1 N Hcl

pH (8.8)

DW _ 250ml

4) Reservoir buffer for Native-PAGE (pH8.3)

 Tris buffer (0.25 M) _ 2.8 g

Glycine (2 M) _ 14.4g

pH (8.3)

Dissolved and made up to 200ml with DW.

5) Sample buffer for Native - PAGE

SGB _ 1.6 ml

Glycerol (optional) _ 2.5ml

Bromophenol blue _ few crystals

Make upto 5ml

6 ) Protein staining solution :

Methanol _ 230 ml

Glacial Acetic Acid _ 50 ml

D/W _ 250ml

Make volume up to 250ml

7) Destaining solution

Methanol _ 75 ml

Glacial acetic acid (10%) _ 25 ml

DW _ 50 ml

Make volume up to 250ml

8) Polymerising agent :

APS 10 % _ 1gm in 10ml

TEMED
Native polyacrylamide gel electrophoresis:

Gel preparation

Resolving gel (10%)

Resolving gel buffer stock _ 7.5 ml

Acrylamide:bis-acrylamide _ 10.00 ml

Ammoniumpersulphate (APS) _ 100l

Water _ 12.3 ml

TEMED _ 50 l

StackingGel (2.5%)

SGS _ 10ml

Ammonium per sulphate (APS) _ 100l

TEMED _ 50 l