Sainfoin trypsin inhibitor : preparation and characterization of the low molecular weight protein
by Walter Frank Baginsky
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biochemistry
Montana State University
© Copyright by Walter Frank Baginsky (1985)
Abstract:
The trypsin inhibitor present in the seeds of the leguminous plant sainfoin (Onobrychis viciifolia,
Scop.), variety Eski, was isolated and biochemically characterized. The inhibitor was isolated by
affinity chromatography on trypsin-Sepharose 4B and the major isoform of the protein was purified to
homogeneity by ion-exchange chromatography on SP-Sephadex C-25. Gel filtration and SDS
electrophoresis showed the inhibitor to be a low molecular weight (6400 daltons) protein and to consist
of a single polypeptide chain of 57 amino acid residues. Amino acid analysis revealed relatively large
amounts of half-cystine (25%), aspartic acid (11%), threonine (11%) and serine (7%) residues. No
sulfhydryl, tryptophanyl, methionyl or carbohydrate components were detected. The amino-terminal
residue of the inhibitor was determined to be half-cystine. Isoelectric focusing showed an isoelectric
point near pH 6.8. The protein was stable to heat, and proteolysis. The inhibitor stoichio-metrically
inhibited bovine trypsin in the molar ratio of 1:1 whereas the inhibition of bovine alpha-chymotrypsin
was weak and non-stoichiometric. Pancreatic elastase and kallikrein were not inhibited by sainfoin
trypsin inhibitor. The purified inhibitor appeared to be an atypical member of the "Bowman-Birk" class
of leguminous protein trypsin inhibitors. SAINFOIN TRYPSIN INHIBITOR:
PREPARATION
AND CHARACTERIZATION OF THE LOW
MOLECULAR WEIGHT PROTEIN
byWalter Frank Baginsky
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Biochemis try
MONTANA STATE UNIVERSITY
Bozeman, Montana
February 1985
APPROVAL
of a thesis submitted by
Walter Frank Baginsky
This thesis has been read by each m e m b e r of the
thesis c o m m i t t e e and has been found to be satisfactory
regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for
submission to the College of Graduate Studies.
vV
Date
Chairpers on
Graduate Commit tee
Approved for the Major Department
/?cP3
Date
Head ,
Approved for the College of Graduate Studies
12 Date
Graduate Dean
ill
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial
fulfillment of
the requirem ent s for a master's degree at Mo nta na State
University,
I
agree
that
the
Library
shall
make
available to borrowers under rules of the Library.
it
Brief
quotations from this thesis are allowable without special
permission,
provided that accurate a c k n o w l e d g e m e n t of
source is made.
Permission
for
extensive
quotation
fr o m
or
reproduction of this thesis may be granted by my major
professor,
or
Libraries when,
use
of
the
in
his
abs e n c e ,
by
the
in the opinion of either,
materia l
is
for
scholarly
Director
of
the proposed
purposes.
Any
copying or use of the material in this thesis (paper) for
financial
permission.
S ignature
gain
shall
not
be
allowed
with out
written
iv
To my Dad
Touchdown
88
V
ACKNOWLEDGMENTS
I would like to express my sincere gratitude
to Dr.
K.D. Hapner for his advice, time and guidance during the
course of this research project.
I wish to ack no wl ed ge Dr. J .E . Robbins and Dr. S.J.
Rogers
for
their greatly
appreciated
discussions
and
support.
I also wish to deeply thank my father for instilling
the desire to succeed and for teaching me so much.
Finally I would like to, thank my wife, Krista, for
her
patience,
understan ding
brings to my life.
and
the
inspiration
she
vi
TABLE OF CONTENTS
Page
LIST OF TABLES......................................
viii
LIST OF FIGURES.....................................
ix
ABSTRACT.............................................
x
INTRODUCTION........................................
I
GOALS AND OBJECTIVES................................
14
MATERIALS AND METHODS..............................
15
Materials. . . ............
Purity Determination of Trypsin and
Alpha-Chymotrypsin............................. . ...
Prbtein Concentrations..........
Purification of Sainfoin Trypsin Isoinhibitors....
Extraction.......................................
Affinity chromatography.........................
I on exchange chromatography.....................
Characterization of Inhibitor 1 1 ..................
Isoelectric focusing............................
Amino acid analysis.......................
Gel filtration...................................
Urea SDS-polyacrylamide gel
;
electrophoresis.............................
Protein transfer from polyacrylamide gel
to nitrocellulose filters..................
Absorption spectrum and extinction
coefficient..................................
Molecular Stability of Inhibitor 1 1 ..............
Resistance toward pepsin........................
Heat treatment...................................
Specificity of Trypsin Inhibitor I I ..............
Determination of Stoichiometry of
Inhibition by Inhibitor II......................... .
Chemical Modification of Inhibitor II .............
Amino-terminal...................................
Carboxymethylation..............................
Performic acid oxidation........................
15
15
16
17
17
17
18
18
18
19
19
19
21
21
22
22
22
22
24
24
24
25
26
vii
TABLE OF CONTENTS— Continued
Page
RESULTS..............................................
27
Purification of Sainfoin Trypsin Isoinhibitors....
Biochemical Characterization of Inhibitor 11 .....
Homogeneity................
Molecular weight...........................
Absorption spectrum and extinction
coefficient...................................
Amino Acid Composition of Isoinhibi tors...........
Molecular Stability of Inhibitor II ...............
Specificity of Inhibitor II........................
Stoichiometry of Inhibition by Inhibitor II ......
Amino-Terminal of Inhibitor II .....................
27
31
31
31
35
37
38
39
39
41
DISCUSSION.............................
43
CONCLUSION.......................
53
REFERENCES CITED
54
viii
LIST OF TABLES
Table
1.
2.
3.
Page
Protein Yields of the Fractions From
the SP-Sephadex C-25 Ion Exchange
Chromatography..............................
30
Amino Acid Composition of the Purified
Trypsin Isoinhibi tors From Sainfoin..........
37
Amino Acid Composition of Alkylated
Inhibitor II .......
42
ix
LIST OF FIGURES
Figure
1.
Page
Affinity chromatography
of sainfoin
trypsin inhibitor . . . ......................
28
Ion exchange chromatography of sainfoin
trypsin inhibitor............................
29
Isoelectric focusing of crude and
purified sainfoin trypsin inhibitors........
32
Urea SDS-polyacrylamide gel electro­
phoresis of crude sainfoin inhibitor
and sainfoin inhibitor II ....................
33
5.
Gel filtration of sainfoin inhibitor Il.....
34
6.
Plot log MW vs_. migration distance
derived from the urea electrophoresis......
36
Stoichiometry of inhibition by sainfoin
trypsin inhibitor.............................
40
Number of amino acid residues within
outermost cystine loop in several
leguminous proteinase inhibitors.............
51
2.
3.
. 4.
7.
8.
X
ABSTRACT
The trypsin inhibitor present in the seeds of the
leguminous plant sainfoin (Onobrychis v'iciifolia, Scop.),
variety
Eski,
was
isolated
and b i o c h e m i c a l l y
characterized.
The inhibitor
was isolated by affinity
ch ro ma t o g r a p h y on try psin-Sepharose 4B and the major
isoform of the protein was purified to hom oge ne it y by
ion-exchange ch ro ma tog ra ph y on SP-Sephadex C - 2 5.
Gel
filtration and SDS electrophoresis showed
the inhibitor
to be a low molecular Weight (6400 daltons) protein and
to consist
of a single polypeptide chain of 57 amino
acid residues.
Amino acid analysis revealed relatively
large amounts of half-cystine (25%), aspartic acid (11%),
threonine (11%) and serine (7%) residues.
No sulfhydryI,
tryptophanyl, methionyl or carbohydrate components were
detected.
The a m i n o - t e r m i n a l residue of the inhibitor
was
determined to be half-cystine.
Isoelectric focusing
showed an isoelectric point near pH 6.8.
The protein was
stable to heat, and proteolysis.
The inhibitor stoichiometrically inhibited bovine trypsin in the molar ratio of
1:1 whereas the inhibition of bovine alpha-chymotrypsin
was weak and non-stoichiometric. Pancreatic elastase and
k a l l i k r e i n w e r e not i n h i b i t e d by s a i n f o i n t r y p s i n
inhibitor.
The purified inhibitor appeared to be an
atypical member of the "Bowman-Birk" class of leguminous
protein trypsin inhibitors.
I
INTRODUCTION
Protein proteinase inhibitors are a diverse group of
proteins
found
throughout
the plant and animal kingdoms.
They possess the ability to associate reversibly with one
or
more
proteinases
protein-protein
functions
[I].
to
form
complexes,
of the proteinases
discrete
sto ichiometric
in w h i c h
all
catalytic
are competitively inhibited
Not only are protein proteinase inhibitors
in number,
but they are also different
toward various proteolytic enzymes;
only one enzyme,
diverse
in specificity
that is, some inhibit
whereas others are polyvalent and can
inhibit several at the same time [2],
For many years proteinase inhibitors have stimulated
the interest of scientists in various
wide
variety
majority
of
inhibitors
of
different
initial
came
from
nutrition,
who
unfavorable
dietary
inhibitors
found
products
[3].
proteinase
work
were
in
Later
reasons.
For
pertaining
thos e
concerned
effects
disciplines,
presented
important
example,
to
involved
about
food
the research
inhibitor-proteinase
animal
potentially
the
plants
emphasis
the
proteinase
with
the
by
for a
proteinase
and
their
turned
toward
interaction,
which
2
offered
procedures
to further
protein interactions,
mechanism
of
basic
[4].
digestion
Members
pharmacological professions
and
of
felt
the
considerable promise
medical
have
been
functions
performed
these
In addition,
to
inhibitors
deduce
what
suggestions
in the
studies
physiological
within
the living
Research in this latter
area has been greatly enhanced by some
speculative
thus having
extensive
might have
tissues of plants and animals.
and
that the inhibitors had
for clinical applications
[5].
the
antigen-antibody
possible potential as therapeutic agents,
field of medicine
protein-
including those which underlie
proteolytic
complexation
understand
intriguing but
[6,7,2,8].
The occurrence of proteinase inhibitors in plants has
been k n o w n since 1 9 38 , when Read and Haas reported that
an aqueous extract of soybean flour inhibited the ability
of trypsin to liquefy gelatins
to its
large
numb er
of
species
is
plant families
regarding proteinase
a legume
as
of
the
a plant
The Leguminosae,
and
significance,
defines
one
[9].
most
their nutritional
extensively
studied
inhibitors.
Webster
characterized
valved seed vessel having a row of seeds
the
seam
These
where
nitrogen
protein-rich
the
parts
fixing
crops,
join,
plants
due
as
are
by a "two-
attached along
in a pod
of peas".
considered
to
be
and offer considerable promise
in
3
supplementing
the protein demands
of susceptible groups.
They have been found to serve a variety of areas which
include:
human
animal feeds.
foods , industrial
In the developing countries,
lack of animal proteins,
balanced
proteins
essential
applications,
amino
aci ds
developing countries,
there is a
or to be more precise,
especially
in
those
the
leguminous
"the meat for poor people" [10].
that
diet
and
of well-
contribute
[10].
In
these
seeds are designated as
The developed countries
have brought modern technology to bear on the isolation
and processing of seed proteins into new foods
[11].
Le gum ino us plant seeds generally account for 20 to
40%
of
the
Furthermore,
total
protein
content
of
the
plant.
they have two to three times as much protein
as cereal grains.
The quantity of lysine was found to be
two to three times higher in legume seeds as opposed to
cereal grain seeds.
This is especially significant since
lysine is the essential amino acid that is most lacking
in diets comprised largely of cereal grains.
Recently,
the
National
Academy
Washi ng to n D.C.,
published a book
that empha si ze d
the importance
resources for the future.
largely
tropics.
unstudied,
of
Sci e n c e s ,
on tropical
of legumes
as
legumes
protein
The book focused mainly on the
potentially
useful
legumes
of
the
The United Nations was also cited as playing a
4
part
through
a "Protein Advisory Group," which
and distributes
concern
scientific
literature
seems feasible
for
much
is expected
of
to be
protein
particularly
becomes more severe,
that in the future,
the
increasingly limited.
mind,
area of
[12].
As the shortage of protein
foods
in this
organizes
the supply of protein
w o rld 's
population
of plant
research
origin
involving
[13].
With
leguminous
the
period
1930-1940,
Moses
inhibitor
established
productive
from
their
cryst allizing
the
the
soybean
protein
inhibitors
complexes,
association.
this
in
plants,
and
n a tur e,
as
well
of
and the
[14].
He
succeeded
as
the
in
trypsin-
investigations
the enz yme -inhi bitor
This work represented
chemical
seeds
pioneered
st oich iom etr y
Kunitz
the naturally occurring
tissue trypsin inhibitor from bovine pancreas,
concerning
be
The majority of available protein
a c c o mp li sh ed the isolation of
inhibitor
will
the seeds, appears to be essential.
During
trypsin
it
the first account of
study on proteinase inhibitors
[15].
Remarkably many of the basic concepts set forth by Kunitz
are
still
proteinase
concepts
retained
in modern
theories
inhibitor interactions.
include
on proteinase-
Examples
1:1 (molar) crystallizable
enzyme and inhibitor;
a quantitative assay
of
these
complexes
of
method for
5
inhibitors;
reversible
complexes at low pH
dissociation
of
enzyme-inhibitor
and the appreciation that the native
co n f o r m a t i o n of the inhibitor is essential in order for
proteinase-inhibitor complexes
Research
on proteinase
to form
inhibitors
period has followed a three step attack.
[I].
since
the Kunitz
Step I consists
of isolation and purification, step 2 deals with research
involving the possible physiological, pharmacological and
nutritional significance of the inhibitors, and step 3,
the most
recent work,
e n z ym e- inh ib ito r
molecular level
involves
interaction
the und ers tanding
and
specificity
at
of
the
[I].
As me nti one d above,
protein proteinase inhibitors
are widely distributed in plants.
The majority of these
inhibitors are found in the seeds, however they are not
necessarily restricted to any one specific part of the
plant.
For
example,
cotyledons were found
in
the
mung
bean
the
leaves
to be high in trypsin
and
inhibitor
activity,
whereas low activity was observed in the stems
and roots
[16].
The seeds of leguminous plants have' long
been perceived as excellent
the
interest
inhibitors
in continued
as well
as
sources of protein.
research
involving
other leguminous
Thus,
soybean
plant proteinase
inhibitors is not surprising.
The
proteolytic
enzymes
fou nd
in
nature
are
6
comprised of four main groups which are characterized by
the
nature
mechanism
of
their
active
involved.
sites
and
the
reaction
These are the serine proteinases,
the sulfhydryl proteinases, the metallo proteinases,
the acidic proteinases.
and
The maj ority of these enzymes
have been shown to be inhibited by proteins isolated from
the
cell s
of
instances,
narrow
plants
and
microorganisms.
the proteinase
inhibitors
span of specificity,
In
demonstrate
some
a very
being able to inhibit one or
possibly two closely related proteinases,
while others of
broad specificity are able to inhibit a much wider range
of
diverse
complexed
include
enzymes
by
[17].
Studies
the proteinase
enzymes
inhibitors
larvae
that often feed on plants and plant products
[18].
These
observations
in
possible
insects
plants also
their
proved
from
from
enzymes
and
the
digestive
involving
to .be very
physiological
important
role
of
plant
studying
proteinase
inhibitors as a defense mechanism against insects.
One
of
deter mi nin g
the
major
ambiguities
encountered
the specificity of a proteinase
in
inhibitor
arises from the fact that some of the early results were
based
on heterogeneous
inhibitory proteins
investigations
on
preparations
[17].
containing
numerous
A large amount of the early
the proteinase
inhibitors
of
plant
origin concentrated almost exclusively on the inhibition
7
of trypsin
[3].
Subsequent studies have shown that many
of the "trypsin
inhibitors"
chymo tryp s in [19].
Some
also
inhibited
inhibitors
the enzyme
of this
type were
shown to contain the same reactive site for both enzymes,
whereas others showed "double-headednes s", the phenomenon
that introduced the concept of two independent inhibitory
sites per molecule of inhibitor.
There are only a few reports available to indicate
that serine proteinase inhibitors also inhibit enzymes of
the other three groups.
One example is the broad bean
inhibitor
trypsin
that inhibits
and chymo trypsin and
also strongly inhibits the sulfhydryl enzyme papain [20].
One key aspect of inhib ito r-enz yme interaction is
the
tight rigidity of the reaction sites.
inhibitor
enzyme
interaction,
or
interaction
inhibitor
appears
key model
[21].
is
ability
their
repla ce me nt
resid ue.
conformational
of
In
substitution
are
Upon enzyme-
changes
negligible.
to represent
in either
The
act ua l
the classical
lock and
One intriguing aspect of the inhibitors
to
the
inhibitory
reactive
so m e
may
retain
site
situations,
lead
to
the
activity
residue
a
by
certain
conversion
of
another
specific
a
strong
trypsin inhibitor to a strong chymotrypsin inhibitor.
most
and
other proteins
substitution
active
of
the
sites
active
are
strongly
site
upon
In
conserved
residue
in
the
8
molecule leads to total loss of activity
The
molecular
weights
of
[7].
plant
proteinase
inhibitors generally fall into two classes,
one with a
comparatively low molecular weight (8,000-10,000) and the
other of
higher molecular
smallest
isolated
c a r b oxypeptidase
mo lecul ar
weight
plant
inhibitor
weight
of
4,300
potato
tubers.
This
One of the
inhibitors
from
potatoes
[22,23].
largest plant inhibitor kno w n
from
(>10,000).
In
is
which
th e
has
contrast,
is the papain
a
the
inhibitor
inhibitor is a glycoprotein
with an estimated molecular weight of 80,000 [24].
Normally,
the leguminous plant proteinase inhibitors
have a molecular weight of approximately 8,000 with high
contents
of
residues.
half-cystine,
In addition,
no tryptophan,
aspartic
inhibitors
free sulfhydryl,
Methonine
is
generally
proteinase
inhibitors
acid
and
serine
of this class
contain
or carbohydrate moieties.
fare.
Larger
leguminous
characteristically have molecular
weight of about 20,000 and low cystine content.
proteins
are
possibly
more
rare,
as
only
the
inhibitors from soybean [25] and winged bean
been
These
trypsin
[26] have
described.
One
of
the
molecular weight
and heat.
most
outstanding
inhibitors
In addition,
is
features
their
of
stability
the low
to acids
many have been shown to be stable
9
to
denatu rat ion
percentage
by
8 M
urea
of half-cystine
solution
residues
confirm extensive cross-linking,
to stability of proteins
leguminous
inhibitor,
[27].
inhibitors,
in
[2].
The
these
large
inhibitors
an important contributor
The high molecular weight
such
as
the K u n i t z
soybean
are low in half-cystine residues and are less
stable as compare d to the "cystine rich" low molecular
weight leguminous inhibitors.
The first plant
was
soybean
inhibitor
trypsin
to be characterized fully
inhibitor
(Kunitz).
Soybean
inhibitor, which has a molecular weight of 22,461, is the
most
studied
inhibitors
of
all
the
legumin ous
plant
proteinase
and has been used as a model for the studies
of other leguminous
proteinase
inhibitors.
The
other
major inhibitor isolated from soybean is the Bowman-Birk
inhibitor that has a molecular weight of 7,975.
molecular
weight
characterized.
It
inhibitor
shows
has
also
double-headedness,
This low
been
fully
inhibits
one
mole each of trypsin and chymo tryp sin simultaneously,
has
seven disulfides, and is resistant to acid, alkali, and
heat.
acid
The Kunitz inhibitor is single-headed,
and heat,
[28,29],
In
proteinase
and
contains
addition
to
inhibitors
only
two
soy b e a n ,
have
characterized from lima bean [30],
unstable to
disulfide
bonds
leguminous
been
plan t
isolated
chick pea [31],
and
winged
10
bean
[26],
garden beans
other leguminous seeds
The
possible
[32],
alfalfa
[33],
and numerous
[34-38].
physiological
role
of
inhibitors is described in reviews by Ryan
recently by Richards on [17].
proteinase
[39] and more
In the broad sense, their
purpose is obvious- inhibition of proteolytic activity.
Nevertheless,
specific functions of the inhibitors are
still unresolved,
particularly so in the case of plant
inhibitors.
The general consensus
of secretory pancreatic
mammals,
on the physiological function
inhibitors,
which occur
in
all
is one of prevention of premature activation of
zymogen activity of the digestive enzymes [40].
It has
also been found that individuals deficient in alpha-1proteinase inhibitor develop pulmonary emphysema rapidly
[41].
lung
E m p h y s e m a is the result of increased turnover of
connective
Research
inhibits
has
tissue
shown
neutrophil
that
proteins,
p rim ar ily
alpha- l-p rot einas e
elastase
than of any other proteinase
at a rate
tested
elastin.
inhibitor
ten-fold
[42].
greater
Additionally,
the large quantities of proteinase inhibitor in mammalian
blood are believed to moderate the reactions leading to
blood clotting
[43].
Several possible roles have been suggested in regard
to the function of inhibitors of plant origin.
Some of
11
the plant proteinase inhibitors have the capability of
inhibiting endogenous proteolytic enzymes of the plant
from
which
protein
they
were
turnover
and
isolated,
thereby
metabolism.
controlling
However,
the
majority
of the plant inhibitors studied apparently do not inhibit
their
own proteinases
suggested
that
the
[39].
proteinase
sulfur depot proteins
large number
feel
that
symbiosis
of cystine
due
with
of a possible
to
Alternatively,
the
Pusztai
inhibitors
may
has
act
as
since many contain a relatively
residues
[8],
leguminous
root-associated
function
of
Other researchers
plants
bacteria,
the
plant
existing
the
in
suggestion
inhibitors
is
to
prevent the plant from being engulfed by the symbiotic
bacteria.
In
addition,
the
inhibitors
may
in
fact
protect the plant tissue at the colonization site against
the action of bacterial proteinases
Recent
possibility
defense
developments
strongly
of plant proteinase
function
against
insect proteinases
[2],
inhibitors
insect
[6,39,44],
emphasize
attack
serving
by
the
as
a
inhibiting
A major advance
in this
field was made in 1972 when Green and Ryan [18] showed
that wound ing of the leaves of potato or tomato plants by
adult Colorado potato beetles
or their larvae produced a
rapid increase
inhibitor
plants tissues.
of proteinase
throughout
the
It was later shown that the accumulation
12
of the inhibitor was directly due to the wounding of the
leaf,
since any type of crushing would cause
induction.
Results of the above study on tomato plants
indicated
the
initiates
the same
probability
the increase
the plant.
Later
of
a
chemical
signal
that
in inhibitor concentration within
research
a substance
showed
produced
that
this
or released
chemical
signal
was
near
the
wound.
The substance in question has been given the name
proteinase inhibitor inducing factor (PIIF), and has been
partially purified
the
release
temperature
with
the
and
[44,45].
transport
dependent
increased
in leaves,
Earlier research proved that
of
[46].
PIIF
is
both
light
These characteristics
accumulation
of proteinase
as the result of insect attack,
and
along
inhibitor
indicate
that
the woun di ng of a single leaf sets off an "immune-like"
response
in
the
plant
[47].
It
is
believed
that
knowledge of this response may play an important role in
the design of new approaches to biological pest control.
Insect
important
pest
and
control
has
controversial
become
one
problems
agricultural community of the world today.
of
the
facing
often
Chemical
control
env i r o n m e n t a l l y
[49,50].
Therefore,
the
In the United
States insects cause estimated annual crop losses
[48].
most
of 15%
of insect pests is expensive and
and
politically
unacceptable
it appears favorable to better our
13
understanding of plant proteinase
inhibitors
in the hope
that ongoing research may one day uncover a biological
"built-in insecticide"
to combat insects.
The use of plant proteinase inhibitors as valuable
laboratory
tools
will continue
to expand.
Researchers
have successfully employed the inhibitors in the area of
affinity
c h r o ma tog ra phy
by
covalently
attaching
the
inhibitor to inert water insoluble pol yme ric supports,
thus purifying specific proteinases
application of plant
proteins
of
was
A second
shown by separating
based on biospecific interaction with a soluble
affinity
[53].
inhibitors
[51,52].
reagent
in conjunction
with
ultrafil tra tion
These findings provide examples of
the, plant
purification
understand
proteinase
tools.
and utilize
inhibitors of plants,
inhibitors
Therefore,
the usefulness
as
in
the capability
isolation
ord e r
of
the
to
and
fully
proteinase
further research is essential.
14
GOALS AND OBJECTIVES
The
principal
bioch em ica l
major
Scop.) .
purification
trypsin
leguminous
litera ture.
and
isoinhibitor
plant
Results
m anu s c r i p t
objective
form
sainfoin
of
the
and
work
of
this
research
is
the
cha rac teriz ation
of
the
from
of
the
the
seeds
(O n o b r y c h is
are
published
viciifolia,
to b e wri tt en
in
the
into
scientific
15
MATERIALS AND METHODS
Materials
Sainfoin seeds (Onobrychis viciifolia, Scop., variety
E s k i ) were
generously supplied by Dr. R.L. D i t t e r l ine,
Montana State University. Alpha-chymotrypsin was obtained
from
Worthington.
Trypsin
pepsin, kailikrein,
elastase,
ethyl ester hydrochloride
tryrosine ethyl ester
4 B , Sephadex G-50,
from Pharmacia.
d e t e rm in ati on
Enzyme
grade
(DPCC
treated,
type
XI),
alpha-N-benz oyI-L-arginine
(BAEE), and
alpha-N-benzoyl-L-
(BTEE) were from Sigma.
Sepharose
and sulfopropyl Sephadex C-25
were
Protein markers for mol ec ular weight
were
urea
purchased
was
from
purchased
Phar macia
from
chemicals were pure or reagent grade.
BRL.
and BRL.
AlI
other
Distilled water
was used throughout.
Purity De termination of Tryps in and Alpha-Chymotrypsin
Trypsin
affinity
used
in
this
ch ro ma tog ra phy
research
on soybean
was
purified
trypsin
by
inhibitor
(STI) that was covalently attached to Sepharose 4B by the
cyanogen
bromide
coupling
method
[54].
A 1.5 x 20 cm
16
column of STI-Sepharose 4B was equilibrated with three
bed volumes
calcium
of .01 M Tris-HCl
chloride
and
.15
M
buffer
sodium
containing
chloride,
.01 M
pH
7.5.
A p p r o x i m a t e l y 25 mg of trypsin (initially dissolved in
.005
M H CI , .005
brought
to
M calcium
chloride
to pH 7.5 with I M Tris-HCl,
the
column
temperature.
buffer,
pH
effluent
at
a fl o w
The
7.5,
rate
column
until
was
the
returned to zero.
280
of
then
nm
and
subsequently
pH 7.5) was applied
20
ml/hr
washed
at
roo m
with
Tris
absorbancy
of
the
Adsorbed trypsin was removed
from the column by elution with .001 M HCl that contained
.01 M calcium chloride and .15 M sodium chloride.
active
fraction was
The functional
then
collected in one test tube at 0°C.
normality
determined
of
by
the collected
active
site
activity
was
analogously
method of Kezdy and Kaiser
Protein
fraction was
titration
p-ni tr ophenyI-p'-gua n idi nob e n z oa te [55].
trypsin
The
wi t h
A l p h a -chy m o -
determi ned
by
the
[56].
Concentrations
Trypsin and alpha-chymo trypsin concentrations
determined
from
their
extinction coefficients
absorbance
[57].
and
were
established
17
Purification o f Sainfoin Tryps in Isoinhibi tors
Ex traction.
Dehulled and
finely ground sainfoin
seeds (280 g) were stirred over night at 4°C in one liter
of pH 7.0, 0.01 M pot ass ium phosphate buffer containing
0.01 M ascorbic acid,
sodium
azide.
material
The
by
0.15 M sodium chloride and .001 M
extract
was
compression
Trichloroacetic acid was
separated from
through
30 min.
cheesecloth.
then added to 2.5% (w/v) and the
extract was stirred one hour at 22°C.
20 min
insoluble
After standing for
the suspension was centrifuged at 10,000 x g for
The supernatant solution was adjusted to pH 7
with 10 M sodium hydroxide.
Affinity c h r o m atography. Tryp sin-S ephar ose 4B was
prepared using cyanogen b r om id e-a ct iva te d Sepharose 4B
and bovine trypsin according to the method of March [54].
The
affinity
matrix
supernatant solution
(10 0
in
ml)
was
added
batchwise fashion,
to stir at room temperature for 2 hours.
affinity matrix was
to
the
and allowed
The adsorbed
collected on a Buchner funnel and
packed
into
a 2.5
X 25.0
cm
washed
with
0.01 M potassium
column.
The
phosphate
colu mn
was
buffer pH 7.0,
containing 0.15 M sodium chloride, until the effluent had
280 nm absorbance
of less than 0.02.
Adsorbed inhibitor
was then eluted from the column with 0.1 M
beta-alanine
18
buffer,
pH 2.5,
that contained
0.15 M sodium chloride.
The trypsin inhibitor fraction was
5 mg/ml
by ultrafiltration
cell using a U M 2
concentrated
in an Amicon ultrafiltration
membrane.
iiLZL e x c h a nge
c h r o m a t o graphy.
The
concentrated
sample was dialyzed in 0.01 M sodium citrate,
then applied
to about
pH 4.0 and
to a 1.5 x 5 0.5 cm column of sulf opropyl
Sephadex C-25, previously equilibrated with pH 4.70, 0.01
M sodium citrate buffer.
Buffer was pumped
column
of
at
peristaltic
a
flow
pump.
rate
The
31
column
ml/hr
was
by
through the
means
developed
column volumes of initial buffer,
with
of
a
two
followed by a linear
gradient of 0.03-0.20 M sodium ion produced from 300 ml
each
of
initial
buffer
and
the
initial
buffer
that
contained 0.17 M sodium chloride.
The colu mn effluent
was
ml
monitored
collected.
desalted
at
Peak
230 nm
and
fractions
by dialysis
against
2.5
were
fractions
individually
water,
were
pooled,
and concentrated by
ultrafiltration over a UM2 membrane.
Characterization o f Inhibi tor 11
Isoelectric
focusing.
Isoelectric
focusing
was
carried out in 0.5 X 10 cm glass tubes, with a Hoefer DE
102
tube gel unit,
according
to the method of Wrigley
19
[58].
Protein
samples
were
focused
acrylamide in a pH 3-10 gradient.
in
6%
total
Bands were visualized
by soaking the gels in 12% (w/v) trichloroacetic acid.
A m ino
acid
analysis.
Ami n o
acid
analyses
were
carried out on a Beckman 120C amino acid analyzer coupled
to an
Infotronics
CRSlIOA
to Spackman et al. [59].
digital
integrator
according
Protein samples were hydrolyzed
with constant boiling HCL in evacuated, sealed hydrolysis
tubes
for 24 hr at IlO0C.
Hydrolyzates were then dried,
dissolved in sodium citrate sample buffer (pH 2.20) and
analyzed.
Gel
filtration.
Molecular-sieve
pe rform ed
on a 0.9 x
fo llowi ng
the method of Andrews
equilibrated
contained
with
0.1
M
1000
cm
colu mn
.05 M Tris-HCl
sodium
chromatography was
chloride
of Sephadex
[60].
G- 5 0
The column was
b u f f e r , pH 7.5,
at a flow
rate
that
of 20
ml/hr at 2 20 C.
Calibration standards were bacitracin
(1,450),
pancreatic
bovine
cytochrome
C (12,400),
trypsin
myoglobin
inhibitor
(17,800)
and
(6,500),
ovalbumin
(70,000).
Urea
SDS- polyacrylamide
gel
electrophoresis.
Urea
SDS-polyacrylamide gel electrophoresis was carried out by
a slight modification of the procedure of Shapiro et al.
20
[61].
Electrophoresis was performed using a
separating gel slab of 15% polyacrylamide
M sodium phosphate pH 7.2,
14 x 16 cm
containing 0.1
0.1% SDS and 6 M ultra pure
urea. ■ The stacking gel consisted of 3.5% polyacrylamide,
and had buffer
gel.
Tank
buffer
that contained
All
conditions
was
identical
0.1 M sodium
to the separating
phosphate,
pH 7.2,
0.1% SD S.
samples
(10 ug) were diluted separately into 40
ul of sample buffer consisting of 0.01 M sodium phosphate
pH 7.2,
7 M urea,
1% SDS, 1% 2-mereap toethanol, and 0.01%
bromp he no l blue.
Prior to application the calibration
standards
were
heated
at
9 5 ° C for
2 min
while
the
inhibitor sample was incubated at room temperature for 15
min.
The gels were run at 5 volts/cm at room temperature
until the bromphenol blue tracking dye reached the bottom
of
the
gel
phoresis
(approximately
was
•solution
containing
isopropanol,
7
hr.
completed,
meth ano l/a ce tic
derived
standards
0.1%
was
ac id/ wat er
weight
from
gels
hr).
After
were placed
Coomassie
el e c t r o ­
in
Blue
staining
G - 2 5 0,
25%
10% acetic acid and 0.1% cupric acetate for
Destaining
molecular
20-22
the
relative
was
performed
24
(5:7:88 by vol).
estimat ed
mob ility
for
of
the
from
in
Inhibitor
standard
reduced
hr
curves
calibration
to the log of their molecular weight.
21
Protein
t r a n sfer
f r om
ni tr ocell ulo se filters.
used
p o l y a c r y l a m i de
The method of Towb in
to elec troelu te proteins
g.£l^ t o ■
[62] was
from po ly acr yl ami de gel
slabs onto nitrocellulose filter paper of 0.2 urn porosity
(Shleicher & Schuell, Keene N H ). The transfer apparatus
was a Hoefer TE series transphor unit,
were
p er for me d
at
buffer (.025 M Tris,
8.3) with
13 ° C .
room
gel
.192 M glycine,
the nitr oce llu lose
gel for 2 hr at a current
transfer,
The
and all transfers
was
transferred
in
20% methanol v/v,
pH
on the anodic side of the
of 1.0 amps.
Fol l o w i n g
the nitrocellulose paper stained for 5 min.
the
at
temperature with 0.2% Amido-Black in 7% acetic acid
and destained
in acetic acid/methanol/water
(7:5:88 v/v).
Absorption spectrum and extinction coefficient.
ultaviolet
absorption
spectrum
of
sainfoin
inhibitor
The
11
was determined using a Varian 635 dual-beam spectrophoto­
meter equipped with a Varian Techtron recorder.
The concentration of a neutral solution of inhibitor
11
was
The
E
I7
determined by
2 8 0 was
quantitative amino acid analysis.
calculated
from
the
concentration
of
protein in the sample from its the absorbance at 2 8 0 nm.
22
Molecular S tability of Inhibi tor 11
Resistance
toward peps in.
The effect of pepsin on
sainfoin inhibitor II was examined.
Inhibitor II was
mixed with pepsin to give a final con cen tration of 5.0
mg/m l inhibitor and 0.5 mg/ml pepsin.
incubated
at
3 70 G.
Aliquots
The mixture was
containing
10
uI
were
removed at various time intervals and mixed separately
with
3
ml
of
immediately
0.05
by
the
M
borate
addition
buffer
of
pH
I ml
9.1,
of
containing .02% fluram (w/v) in acetone.
followed
a solution
Fluorescence
was determined with the Varian instrument using exciting
light at 400 nm.
As a control,
bovine serum albumin was
treated under identical conditions.
Heat
treatment.
Heat stability of inhibitor II was
determined by the following method.
The inhibitor sample
was
At
incubated
intervals,
at
9 50 C for
aliquots
of
3 hr.
the
inhibitor
increasing
time
solution
were
withdrawn and assayed for trypsin inhibition.
II incubated at 22°C
Inhibitor
was used as a control.
Specificity of Trypsin Inhibitor II
Trypsin and ch ymo tr yp sin esterase activity
measured
were
spectrophotometrically using enzyme rate assays
23
described by Walsh and Wilcox [57].
carried
out
concentrations
at
room
All experiments were
temperature.
The
substrate
used for the inhibitory assays
were
5.25
X I 0 ~ ^ M a Ip h a - N -ben z oy I- L - a rginine ethyl ester (BAEE)
and
2.91 X IO-^ M alpha-N-benzoyl-L-tyrosine ethyl ester
(BTEE).
Elastase and kailikrein activity were
determined
in a similar fashion utilizing substrates N-benzoyl-Lalanine- m e thyl ester and BAEE,
respectively
[63,64].
The inhibitory activity of sainfoin inhibitor II was
obtained from the change in slope as recorded spectroph ot om et r i c a l l y
from
containing enzyme
reaction
an
mixture
aliquot
of
a
series
and varied
involving
inhibitor
of
reaction
amounts
trypsin
(0-45
mixtures
of inhibitor.
inhibition
u I containing
The
contained
0-21
ug
protein) diluted to 200 uI with 0.05 M T r i s - H C 1 , 0.01 M
calcium chloride,
uI
(7
0.15 M sodium chloride,
u g ) trypsin.
The
chy mot ry ps in
incorporated an aliquot of inhibitor
pH 7.80 and 20
activity
assay
(0-65 ul containing
0-30 ug protein) diluted to 200 ul with 0.05 M Tris-HCl,
0.01 M calcium chloride,
and
0.15 M sodium chloride,
10 ul (7 ug) alpha-chymotrypsin.
Reaction solutions
were allowed to incubate 10 min at room
this time, 100 ul aliquots
using the appropriate
pH 7.8
temperature.
At
were w i t h d r a w n and assayed
substrates.
24
Determination of Stoichiometry of Inhibition by Inhibi tor
II
Purified
sainfoin
trypsin
inhibitor
II
was
adjusted to pH 7.5 and repurified on a 0.5 x 3 cm c oIumn
of trypsin-Sepharose 4B in order to assure 100% active
inhibitor for use in the sto ich iomet ry
determination.
The column was equilibrated at room temperature with two
bed volumes
of pH 7.5,
.01 M Tris-HCl
application of inhibitor sample.
buffer
prior
The column was
to
then
washed with 10 volumes of the same buffer fol lowed by
release
of
the
active
inhibitor
upon
elution
with
I m M HC1.
Data points obtained from the enzyme rate assay of
inhibited enzyme
were
treated by least
squares
analysis
which resulted in a x-i ntercept value indicative of the
quantity of inhibitor which inhibits 100% of enzyme.
stoic hi om etr y
The
of inhibition is then determi ned by the
molar ratio of inhibitor to enzyme at the point of 100%
inhibition.
Chemical Modification of Inhibitor II
A m in o - t e r m inal.
sainfoin
similar
inhibitor
to
th at
The
II
amino-terminal
was
of 1
N amen
deduced
and
Hapn e r
by
a
[65].
residue
of
procedure
A
sample
25
containing .45 mg of inhibitor II was dialyzed against
0.1 M sodium bicarbonate pH 8.4 for 5 hr.
a 40-fold
molar
excess
of
amino groups was added.
8.4 and
incubated
iodoacetic
After dialysis
acid
over
total
The solution was adjusted to pH
at 5 4 ° C for
19 hr.
Excess
reagents
were removed by dialyzing against water for 5 hr.
inhibitor
sample
was
then
evaporated
to
The
dryness,
hydrolyzed with I ml of 6 N HCl at IlO0C for 24 hr and
analyzed on the amino acid analyzer.
The results were
compared
to
of
inhibitor
sample
of
an a m i n o
the
amino
not
acid
acid
treated
residue
analysis
with
was
a comparable
iodoacetic
assumed
acid.
to
be
Loss
due
to
alkylation of the nitrogen-terminus.
Carboxymethylation.
me t h y l a t i o n
according
to
lyophilized
of
the
The
reduction
sainfoin
inhibitor
method
of Thomas
protein
was
11
and
was
et a I.
dissolved
carboxyperformed
[66].
in
a
The
solution
consisting of 6 M guanidinium chloride and 0.1 M Tris, pH
9.5.
Reducti on was carried out by the addition of a 10-
fold molar excess of dithiothYeotol.
incubated
under nitrogen,
The solution was
in the dark,
for
5 hr.
The
inhibitor was then carboxyme thyIa te d with a 3-fold molar
excess
of
recrystallized
was quenched after
iodoacetic
acid.
The
reaction
5 min by the addition of a 10-fold
molar excess of 2-mercaptoethanol.
Excess reagents were
26
removed by dialysis in water at 4°C.
carboxymethylated
After dialysis,
the
inhibitor was lyophilized, hydrolyzed,
and analyzed for S - c a r b ox ym eth yl cys te ine with an amino
acid
analyzer.
Pe r f o rmic
a c id
inhibitor
was
subjected
to amino acid analysis
cysteic
acid.
oxidized
oxidation.
the
Sainfoin
method
of Hirs
trypsin
[67]
and
for det erm in at ion of
27
RESULTS
Purification of Sainfoin Trypsin Isoinhibitors
Fig. I illustrates the elution profile obtained by
affinity
chromatography
of
the initial
crude
extract
on
tryps in-Sepharose 4B. Equilibrating, buffer was used to
wash nonads orbed inactive proteins and pigments from the
column.
Upon
the
addition
of
the pH 2.5 buffer,
dissociated
fr o m
t he
the
adsorbed
inhibitors
trypsin-
Sepharose
4B and eluted as a sharp peak of absorbancy.
BAEE assay of collected fractions showed no inhibitory
activity
peak
prior
to the addition
containing
concentrated.
the
active
of pH 2.5 buffer.
protein
was
The
pooled
and
The buffer salts were removed by dialysis
of the active fraction in 0.01 M sodium citrate,
pH 4.0.
The dialyzed sample was subjected to ion-exchange
ch rom ato gr ap hy on s u l f o pro py I-Sephadex G - 2 5 at pH 4.70.
As
shown
in
obtained.
Fig.
2,
four
peaks
of
Peaks I and 2 emerged
absorbancy
were
from the col umn with
initial buffer, while peaks 3 and 4 were eluted with an
increasing
gradient
isoinhibitor
NaCl
and
(peak
showed
of
sodium
3) eluted
affinity
for
chloride.
The
major
at app rox im at ely
0.08 M
the
ion
exchange
resin
u 0.60
ELUTION
Figure I.
VOLUME
(ml)
Affinity chromatography of sainfoin trypsin
inhibitor.
Elution of crude sainfoin trypsin
inhibitor is with 0.1 M beta-alanine (arrow).
Na
3
CO 0 .5'
125
FRACTION
Figure 2.
Ion exchange chromatography of sainfoin
trypsin inhibitor.
Fractions indicated
by number were pooled.
30
intermediate of that of peaks 2 and 4.
showed
strong
inhibitory
activity
Peaks 2, 3, and 4
against
trypsin,
whereas fraction I, not retained by the column,
only trace amounts of inhibitory activity.
pooled
as
indicated
Pe a k s
2,
3,
and
and
showed
Each peak was
concentrated by ultrafiltration.
4 were
designated
as
the
trypsin
inhibitor
fractions
yields of
the individually pooled peaks obtained from
the SP-Sephadex
in Table. I.
abundant as
C-25
The
I, II and III, respectively.
The
ion exchange column are summarized
major
isoinhibitor
11
was
twice
the other isoforms and represented about 35%
of total crude inhibitor.
Table I.
as
Yields of the Peaks From the SP-Sephadex C-25
Ion Exchange Chromatography.
Protein
(mgs )
Yielda
(%)
I
2.8
19.4
2
0.8
5.6
3
5 .I
35.4
4
2.6
18. I
11.3
78.5
Peak
Total
aBased on total protein applied to the column.
31
Biochemical Characterization of Inhibi tor 11
Homogeneity.
Determination of purity was performed
by isoelectric focusing and the results are shown in Fig.
3.
Inhibitor
II
(major
isoform)
produced
a
single
homogeneous band that focused near pH 6.8 (estimated from
calibration
inhibitor
protein,
II,
homogeneous,
not
shown).
inhibitors
and
focused
respectively.
Further
homogeneous
of
state
I
In
an d
near
pH
supporting
sainfoin
inhibitor
addition
III
8.1
were
and
data
11 was
by urea SDS-polyacrylamide gel electrophoresis.
shows
band
the results.
near
the
to
also
pH
4.3,
for
the
obtained
Figure 4
Inhibitor II migrated as a single
position
of
bovine
pancreatic
trypsin
inhibitor which has a molecular weight of 6,500.
If the
inhibitor II was heated prior to application to the gel
slab, smeared and diffuse staining occurred indicating
molecular
degradation.
Molecular weight.
The molecular weight of inhibitor
II determined by gel filtration on a calibrated column of
of
Sephadex
Fig.
5.
identical
The
G- 5 0
was
found
inhibitor
elution
time
inhibitor (M.¥. 6,500).
to
eluted
as
be
as
bovine
6,500
as
a single
shown
peak
pancreatic
in
with
trypsin
Amino acid analysis (see later)
32
1
Figure 3.
2
3
4
Isoelectric focusing of crude and purified
sainfoin trypsin inhibitors.
Gel I, crude
inhibitor; gel 2, inhibitor II; gel 3 ,
inhibitor III; gel 4, inhibitor I .
33
A
Figure 4.
B
C
D
E
Urea SDS-poylacrylamide gel electrophoresis
of crude sainfoin inhibitor and sainfoin
inhibitor II.
Lane A, control; lane B ,
molecular weight markers ; lane C , crude
sainfoin inhibitor; lane D , sainfoin
inhibitor II; lane E , bovine pancreatic
trypsin inhibitor.
Bacitracin
2 .2 -
Ve/Vo
1.8 B o v i n e P a n c r e a t i c Trypsin Inhi bi t or
Cytochrome C
1.4 Myoglobin
1.0+i
3.0
3.4
3.8
4.2
4.6
5.0
LOG MW
Figure 5
Gel filtration of sainfoin inhibitor II.
Arrow indicates elution volume of inhibitor II.
35
showed inhibitor II to have a minimal molecular weight of
6,384.
Inhibitor
weight
II was
de te rm ina ti on
electrophoresis.
migrated
as
The
a
further analyzed for molecular
by
urea
reduced
S D S-p olya cry lamid e
and denatured
homogeneous
spe cies.
inhibitor
The
single
homogeneous band representing inhibitor II migrated
a
mobil ity
comparable
to
that
of
trypsin inhibitor (Fig. 4, Lane C).
bovine
gel
with
pancreatic
The molecular weight
value of inhibitor II was esti mated from a plot of log
molecular
standard
weight
marker
observation
phoretic
vs
of
protein
proteins
a
single
conditions
migration
(Fig.
band
6).
under
indicated
established
with
Furthermore,
reduced
that
the
e l ect ro ­
inhibitor
11
consisted of a single polypeptide chain.
Absorption spectrum and extinction coefficient.
ultraviolet absorption spectrum
The
of sainfoin inhibitor II
in 0.01 M sodium citrate, 0.1 M sodium chloride, pH 4.70
showed a m a x i m u m at 27 5 nm and a high m i n i m u m at 260 nm,
indicative
of
the
amount
large
the
tyrosine
of
chr o.mophore as
h a l f - c y s tine.
influenced
The
by
extinction
coefficient E i* was calculated to be 7.92 for inhibitor
II at 280 nm.
Ovalbumin
-Chym otrypsinogen
B- Lact o g I o b u l in
L y s o z y m e / C y to c h r o m e C
B o v i ne P a n c r e a t i c Trypsin I nhi bi t or
Insul i n
4-0
MIGRATION
Figure 6.
6-0
80
DISTANCE (cm)
Plot log MW v_^. migration distance derived
from the urea electrophoresis.
Sainfoin
inhibitor II (arrow).
37
Amino Ac Id Composition of Isoinhibi tors
Results of amino acid analysis of sainfoin inhibitor
are shown in Table 2. The major isoinhibitor II protein
contained 57 total amino acid residues.
Table 2
Half-cystine,
Amino Acid Composition of the Purified Trypsin
Isoinhibitors From Sainfoin.a ’
Amino Acid
Inhibi tor
I
Lysine
Histidine
Arginine
Aspartic Acid
Threonine
Serine
Glutamic Acid
Proline
Glycine
Alanine
Half-Cys tine
V aline
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Tryptophanc
Total
Molecular weight
3.4
1.0
3.0
6.0
4.7
3.6
4.9
I .I
2.6
4.0
9.7
2.0
0.0
2.7
2.6
I .6
2.0
0.0
(3)
(I)
(3)
(6)
(5)
(4)
(5)
(I)
(3)
(4)
(10)
(2)
(0)
(3)
(3)
(2)
(2)
(0)
57
6384
Inhibi tor
II
2 .I
1.0
3.2
6.0
6.0
3.8
3.2
4.1
I .I
3.0
13.3
0.8
0.0
2.6
I .8
1.7
I .8
0.0
(2)
(I)
(3)
(6)
(6)
(4)
(3)
(4)
(I)
(3)
(14)
(I)
(0)
(3)
(2) .
(2)
(2)
(0)
57
6384
Inhibi tor
III
3.3
(3)
1.0 (I)
3.8
(4)
6.0
(6)
7.3
(7)
5.0
(5)
3.3
(3)
4.7
(5)
I .3 (I)
3.4
(3)
14.6 (14)
1 .5
(2)
0.0
(0)
2.9
(3)
I .8 (2)
1.8
(2)
I .8 (2)
0.0
(0)
63
7056
aAll values quoted are residues/molecule. ^Nearest
integers are
shown in parentheses.
cValue
determined by UV spectrum analysis.
.
38
aspartic acid, threonine and serine were present in large
amounts and represented 53% of all amino acid residues.
No
tryptophan,
methionine,
was observed. Co mpa rab le
galactosa m ine, or glucosamine
amino acid com positions were
shown for inhibitors I and III (Table 2).
acid
the
analysis
absence
of
of
alkylated
native
In the amino
sainfoin
carboxymethylcysteine
inhibitor,
indicated
free sulfhydryl groups were present,
suggesting
the
in
half-cystine
residues
Subsequent analysis
existed
of denatured,
sainfoin inhibitor resulted
inhibitor,
that all
disulfide
reduced,
form.
and alkylated
in the recovery of 12.4 umol
carb o x y m e thyI c y s t e i n e /umoI
sainfoin
that no
protein,
according
whereas
oxidized
to amino acid analysis,
gave a value of 10.65 umol cysteic acid/umol protein.
Molecular S tability of Inhibi tor 11
The
II
was
conformational
investigated
stability
by exposure
samples to heat treatment,
inhibitor
its
II appeared
ability to inhibit
of
sainfoin
of various
trypsin, and pepsin.
to maintain
inhibitor
inhibitor
Sainfoin
100% effectiveness
in
trypsin after exposure to pepsin
or after treatment with 2.5% trichloroacetic acid for I
hr at room temperature. No inhibitory activity was lost
when the inhibitor was placed in a boiling water bath at
9 5 0C for 3 hr.
39
Specific!ty of Inhibi tor 11
The
inhibitor
strongly
inhibited
inhibition of ch ymo tr yps in was
weak.
trypsin whereas
In addition,
no
inhibitory activity was exhibited by inhibitor II against
the
animal
pancreatic
against kallikrein,
serine
proteinase
elastase
nor
a trypsin-like enzyme from porcine
pancreas.
;
Stoichiometry of Inhibition by Inhibi tor 11
The activity of bovine trypsin and chymotrypsin was
determined to be 7 6% and 79%, respectively.
From these
values the amount (mg) of active enzyme pre.sent in the
inhibitory
assays
inhibitory
activity
bovine
trypsin
inhibitor
bovine
calculated.
of sainfoin
and
II was
trypsin,
was
Fig.
inhibitor
7,
linear
the
to
shown
whereas
inhibition of
indicating
inhibition
about
20%
II
alpha-chymotrypsin.
the inhibition of
of
trypsin
residual
by
against
inhibit
chyomotrypsin
As shown in
inhibitor
activity,
the
Sainfoin
to s t oic hi ome tr ica ll y
was found to be weak and non-stoichiometric.
Fig.
7 shows
II is
whereas
the
chymotrypsin was limited and non-linear
relative
linear portion of
weak
binding.
Extrapolation
the trypsin-inhibitor curve
of
the
to zero
enzymatic activity indicated the inhibitor (mg) necessary
Mol e I n h i b i t o r / M o l e E n z y m e
Figure 7.
Stoichiometry of inhibition by sainfoin
trypsin inhibitor.
Inhibition of bovine
trypsin by inhibitor II (major isoform)
★ - - - ★ . Inhibition of bovine a Ipha chymotrypsin by inhibitor 11
■- - - ■
41
for compl ete
inhibition of the enzyme.
Calculation of
the molar conecentrations of both inhibitor and enzyme at
zero enzyme
activity resulted in bindingstoichiom etry of
1 :1 .
Amino-Terminal Determ!nation of Inhibi tor 11
As
shown, in
Table
3,
amino
acid
analysis
of
alkylated inhibitor II indicated, the loss of a cystine
residue
native
when
compared
inhibitor
II.
to
the
All
amino
other
acid
amino
analysis
acid
of
residues
remained unchanged with the exception of glycine which
increased from one to two residues.
This increase was
attributed
of
to
degradatory
by-products
reaction and has been observed elsewhere
the
alkylation
[65].
The loss of cystine was credited to the alkylation
of its fr.ee alpha amino group
cystine was N-terminal.
thereby concluding that
If alkylation of inhibitor II
was performed at room temperature, no apparent alkylation
was observed (data not shown), possibly indicating that
the amino-terminus was resistant to alkylation.
42
Table 3
Amino Acid C om po sit io n of Alkylated
Inhibitor II.a ’b
Lysine
MCMLc
His tidine
Arginine
Aspartic Acid
Threonine
Serine
Glutamic Acid
Proline
Glycine
Alanine
Half-Cys tine
Valine
M e thionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Tryp tophan
I .O (I)
I.O (I)
O .9 (I)
3.0
(3)
6 .O (6)
5.7
(6)
4.0
(4)
3.4
(3)
4.0
(4)
1.8
(2)
2.9
(3)
11.1 (12)
0.9
(I)
0.0
(0)
(3)
2.8
2.0
(2)
I .6 (2)
1.8
(2)
(0)
O
Alkylated
Inhibitor
II
O
Amino Acid
Non-Alkylated
Inhibi tor
II
2 .I
0.0
I .0
3.2
6.0
6.0
3.8
3.2
4 .I
I .I
3.0
13.3
0.8
0.0
2.6
1.8
I .7
1.8
0.0
(2)
(0)
(I)
(3)
(6)
(6)
(4)
(3)
(4)
(I)
(3)
(14)
(I)
(P)
- (3)
(2)
(2)
(2)
(0)
a All values quoted are residues/molecule.
bNearest integers are shown in parentheses,
c monocarboxymethyllysine.
43
DISCUSSION
This thesis reports on exp eri me nt al findings that
show
that the seeds from
sainfoin
the perennial
leguminous
contain a highly stable proteinase
plant
inhibitor
with primary inhibitory activity directed toward trypsin.
It was
shown that all trypsin inhibitory activity was
removed from the sainfoin seed extract with a single pass
through an affinity ch romat og rap hy column of trypsinSepharose 4B, thereby separating essentially all of the
sainfoin
trypsin
inhibitors
from
other soluble proteins.
As shown in Figure I, some peak tailing occurred during
elution of the inhibitor from the affinity column.
cause of the peak tailing is unclear, although
The
it may be
due to variable affinity b e twe en the inhibitor a-nd the
trypsin matrix or perhaps a result of the positioning of
the binding sites on the resin rendering some sites less
accessible
purification,
than
others.
Following
this
method
of
the amount of trypsin inhibitors obtainable
from sainfoin seeds was determined
to be 60 mg/Kg.
This
value is low in comparison with the yields of other known
small molecular weight plant proteinase inhibitors
such
as mung bean which contains 250 mg of inhibitor per Kg of
seeds
[31].
Consequently,
sainfoin
seeds
appear
to
44
contain the lowe st source of free unbound inhibitors as
compared to other leguminous seeds.
The
acid
omission
step
in
or utilization
the
extraction
of
the
trichloroacetic
procedure
no
detectable
difference
inhibitor.
From this observation, it thus appears that
the
stability
of
in yield values
showed
the
sainfoin
of the purified
inhibitor
toward
trichloroacetic acid (TCA) treatment enables the use of
T CA
as
an
efficient
means
of
precipitating
insoluble material out of the extract,
the
unwanted
without affecting
inhibitor.
The
heterogeneous
mixture
of
inhibitors
was
successfully separated using ion exchange chromatography
on
suIfopropyl-Sephadex
C- 25
(Fig.
preparations were chr oma to gra ph ed
were
found
to
profiles,
obtaining
thus
form.
the
purification
a
inhibitor
If
higher
previously
procedures
identical
reproducible
II
(major
yields
mentioned
can
Numerous
in this fashion and
essentially
assuring
sainfoin
ho mog eneou s
neede d,
exhibit
2).
be
elution
means
isoform)
of
in a
inhibitor
isolation
modified
in
of
order
are
and
to
accommodate the inhibitor demand.
The
total
yield
of
protein
isolated
from
the
aforementioned ion exchange chromatography was estimated
at
7 8.5%
based
on
total
protein
applied
to
the
ion
45
exchange resin (Table I).
Therefore it was of interest
to inquire whether any or all of the remaining 22.5% was
still bound to the ion exchange resin.
It was assumed
that if additional protein was present on the column,
then a more concentrated salt solution (in comparison to
the
applied
elution.
the
salt
gradient)
was
necessary
to
enhance
In order to confirm or deny this assumption,
following
procedure
was
followed.
Once
the
chromatography had reached a salt gradient concentration
of .2 M , a salt solution of sodium chloride
passed
through
the ion exchange resin.
(.5 M ) was
This resulted in
the elution of a protein fraction having an estimated
yield
value
strong
of
2.2
mg.
inhibitory
additional
experiment
The
protein
activity
fraction
against
utilizing
urea
showed
try ps in .
An
SDS-polyacrylamide
gel electrophoresis resulted in the observation of one
intense
band and
two lighter bands.
The intense band
migrated identically to that of the 22,500 dal ton band of
the crude
inhibitor which is shown in Fig.
Although
separation
ho mogen eou s
components
characterization,
of
a
high
of
this
is
it appears
mol ecular
weight
protein
necessary
possible
trypsin
contained within the seeds of sainfoin.
4 (lane B ).
fraction
for
that
into
additional
the
presence
inhibitor
maybe
The conformation
of a sainfoin inhibitor of this type would result, in one
46
of the few high molecular weight species isolated from a
leguminous
plant.
On the basis of inhibitory activity against trypsin,
and amino
acid
com po si ti on
data,
the elution
profile
shown in Fig. 2
suggests the presence of three trypsin
isoinhibitors
in
sainfoin
seeds.
According
to
isoelectric focusing results all three isoinhibitors were
obtained
in
a
homogeneous
state
(Fig.
3).
The
observation of more than one isoinhibitor contained in
the
seeds
of
a leguminous
plant
is
not
an
unc o m m o n
occurrence, as analogous findings have been documented
for
a
wide
Sainfoin
variety
of
inhibitor
II
incubated
reduced
at
9 5 °G
inhibitor
for
II
leguminous
was
3
found
h r.
to be
Although
9 50 G
at
seeds
[26,33,68].
stable
when
incubation
resulted
in
of
molecular
degradation as observed on urea SD S- p o Iya cr y I a mide gel
electrophoresis.
appears
to
disulfide
The instability of reduced inhibitor II
strongly
bonds
in
support
pH
conditions
importance
maintaining
architecture of the protein.
acidic
the
of
the
of
the
structural
Pepsin treatment as well as
the
purified
inhibitor
were
without effect on the inhibitory activity of the protein.
In regard to the possibility of utilizing sainfoin as a
possible forage crop,
these stability
characteristics
were examined from a nutritive aspect. - The resistance of
inhibitor II to peptic digestion and acidic conditions is
significantly
relevant
from
a nutritional
standpoint
since ingested sainfoin inhibitor 11 by livestock will
probably pass
through
the
stomach
unaffected.
However,
possible adverse physiological effects are unlikely since
weanl ing rats fed on a sainfoin diet showed no sign of
pancreatic hypertrophy
All
previously
[69].
studied
leguminous
plant
proteinase
inhibitors consist of two tandem homology regions on the
same
polypeptide
chain,
each
with
thereby stoichiometrically inhibiting
per I mole of inhibitor.
a
reactive
2
moles of enzyme
Functionally,
site,
inhibitors are
termed as single-headed if they have only one reactive
site on the molecule
Fig.
6,
shows
[70].
that
inhibitor
II
comp lexed
with
trypsin on a 1:1 molar basis, whereas the inhibition of
chymotrypsin was found to be weak and non-stoichiometric.
In
order
to
attain
these
inhibitory
results
it
Was
essential that sainfoin inhibitor II be repurified over a
column
of
trypsin-Sepharose
repurif ication
relationship.
step
resulted
The reason as
4B .
in
Omitting
a different
to the presence
this
molar
of inactive
material contained within the purified sainfoin inhibitor
II sample
remains
unknown.
One
seemingly
reasonable
rationale for the occurrence of the inactive substance
48
may
be
partly
ascribed
inhibitor
during
inhibitor
is
to partial
purification,
purified
chromatography.
by
proteolysis
especially
methods
of the
when
involving
the
affinity
These findings suggest that inhibitor II
is comprised of a single inhibitory site per molecule and
moreo ver
indicates
the
first
doc um ent at ion
of
a low
molecular weight leguminous plant inhibitor showing high
specificity
ratio
of
for a single
1:1.
enzyme
Therefore,
resulting
these
sainfoin inhibitor II represents
findings
in a molar
suggest
that
the first instance of a
"true" single-headed inhibitor isolated from a leguminous
plant.
Inhibitor II can be classified as a member of the
Bowman-Birk
family
molecular weight,
and
on the basis
of low
high contents of aspartic acid,
serine
half-cystine
carbohydrate
of inhibitors
and
moeity.
the
absence
However,
the
of
tryptophan
inhibitor
and
contains
a
unique type of single-headedness highly specific for a
single
proteinase.
the existence
molec ula r
This
inhibitory
of a Bowman-Birk
forms
showing
phenomenon
sub-family consisting of
"single-headed"
In addition to inhibitory "uniqueness",
appears
among
to have
Bowman-Birk
inhibitors.
the
the lowest
lowe st
type
suggests
recorded
leguminous
inhibition.
inhibitor II also
mole cular
plant
weight
proteinase
The occurrence of inhibitor II possessing
molecul ar
weight
along
with
single headed
49
nature poses some interesting questions.
One
possible
explanation
of
nature of inhibitor 11 and its low
lie
in
the
Initially,
pre paration
and
the single
molecular
pur ification
weight mayprocedures.
the sainfoin extract was per mit ted
overnight at pH 7.0.
conditions
headed
to stir
It appears quite feasible that at
of neutral pH,
endogenous proteases
contained
in the extract may cleave susceptible peptide bonds of
the
inhibitor
content
of
molecule.
disulfide
Due
bonds
protein in its native state,
to proteolytic attack.
to
the
(Table
inhibitors'
2),
the
high
purified
is virtually 100% resistant
It appears
that if cleavage does
occur the probable site of proteolytic attack on native
sainfoin inhibitor II would be the amino acid residues
terminal to the
external most cystine residue.
If it is assumed
that peptide
bond cleavage does
occur at these positions and the postulated carboxyl and
nitrogen
terminal peptides are omitted from the amino
acid composition,
then what remains is the body of the
inhibitor which will be referred to as the "core protein"
(Fig. 8).
In comparison of the amino acid composition of
sainfoin inhibitor II with the core protein amino acid
composition of Bowman-Birk soybean trypsin inhibitor and
Garden Bean inhibitor
compositions
and
II, almo st
molecular
weights
identical amino acid
were
observed.
This
50
adds
support
to
the
possibility
of
the
isolation
of
sainfoin inhibitor II as a protein devoid of its nitrogen
and
carboxyl
terminal
amino
outermost cystine residue.
aci d s
external
to
the
Furthermore, comp ariso n of
the amino acid sequence of the proteinase inhibitors from
garden
bean,
degree
of
cores
Bow man-Birk,
structural
[17].
and
lima
hom ol ogy
bean
with
show
their
a high
respective
These regions of internal homology contain
the reactive
site(s)
of the inhibitor which
interacts
with the appropriate enzyme (s).
As mention ed earlier, sainfoin inhibitor II is the
first reported occurrence of a single-headed inhibitor
capable of inhibiting only one enzyme at a molar ratio of
1:1.
It appears
flanking
conceivable
nitrogen
and
that
carboxyl
if
cleavage
terminal
of
amino
the
acid
residues external to the outermost cystine residue of the
"core protein" has occurred,
then their absence may have
disrupted
the
second
inhibitor
II.
potential
In
support
of
reactive
this
site
hypothesis,
observation of a C-terminal residue being part
on
the
of the
reactive site of an inhibitor was reported by Fritz [70].
Additionally,
of
several
the molecular weight
leguminous
inhibitors
to that of inhibitor II.
of the core proteins
are reasonably
similar
S oybean
Lima Bean
Garden Bean
Peanut
Mung Bean
Adzuki
55
58
55
58
55
55
S-S
Sainfoin 57
Figure 8.
-S-S
Number of amino acid residues within outermost
cystine loop in several leguminous proteinase
inhibitors.
52
Further
research
regarding
determination
and acidic
pH
preparations
is in progress.
primary
conditions
during
structure
inhibitor
Results of this research
will hopefully resolve the question as to whether or not
sainfoin inhibitor II has been modified by the action of
endogenous proteinases.
will
lend
insight
in
Additionally,
the
the sequence work
det erm in at ion
second reactive site on inhibitor II.
of a possible
53
CONCLUSIONS
1.
Sainfoin seeds contain a stable low molecular weight
proteinase inhibitor belonging to the Bowman-Birk
class.
Typically, 60 mg of crude inhibitor was
isolated from I Kg of seeds.
2.
Inhibitor II was purified to homogeneity by affinity
and ion exchange chromatography.
Homogeneity was
confirmed by isoelectric- focusing, electrophoresis
and amino acid analysis.
3.
The molecular structure of purified inhibitor II
consisted of a single polypeptide chain composed
of 57 amino acid residues.
The molecule had 7
disulfide bonds and calculated molecular weight
of 6,384.
4.
Inhibitor II was specific for trypsin and showed a
stoichiometric molar binding ratio of 1:1.
5.
Unusual apsects of inhibitor II are its low molecular
weight and its apparent single-headed nature.
54
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Laskowski, M . Jr. and Sealock, R .W . 1970.
In:
The
Enzymes , P . Boyer, Ed., Academic Press, New York.
3:375-473.
2.
Vogel, R., Trautschold, I . and Werle, E . 1968.
Natural Pro teinase Inhibi tors. Academic Press,
New York, pp. 9-42.
3.
Pusztai, A.
1967.
Trypsin Inhibitors of Plant
Origin, Their Chemistry and Potential Role in Animal
Nutrition.
Nutr. Abs t. R e v . 37:1-9.
4.
Kowalski, D . and Laskowski, M., Jr.
1972.
Inactiva­
tion of Enzymatically Modified Trypsin Inhibitors
Upon ChjBmical Modification of the Alpha-Amino Group
in the Reactive Site.
Biochemistry.
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5.
Werle, E . 1971.
In:
Proceedings of the 1st
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Walter deGruyter, Berlin.
pp. 23-27.
6.
Ryan, C .A . 1983.
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Variable Plants and
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Laskowski, M., Jr. and Ka to , I . 1980.
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8.
Pusztai, A.
1972.
Metabolism of TrypsinInhibitory Proteins in the Germinating Seeds of
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9.
Read, J.W. and Haas, L .W . 1938.
Studies on the
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Actions.
V. Further Studies Concerning Potassium
Bromate and Enzyme Activity.
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Mosse , J . and Pernollet, J .C . 1983 . In:
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11.
Lillford, P .J . 1978 . In:
Plant Proteins, G .
Norton, E d . , Butterworths, London, pp. 289-298.
12.
In:
Chemistry and Biochemistry
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MONTANA STATE UNIVERSITY LIBRARIES
762
1001 1 9 8 5 6
KAT?
N370 Baginsky, W. F.
B145
Sainfoin trypsin
cop.2 inhibitor
I S SUED T O
WATTT
N378
Blk 5
cop. 2