ENHANCED SELECTIVITY OF A MULTIFUNCTIONAL BIOCATALYST, PLASMIN by
PATRICIA 0. ZUNIGA, B.S. A THESIS IN CHEMICAL ENGINEERING Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE IN CHEMICAL ENGINEERING Approved
Accepted
May, 1985
ACKNOWLEDGEMENTS I would
like
to
thank my family for their support,
patience, and understanding over the past three years. I would also like to thank the members of my committee for their encouragement and support. A special thanks is extended to Dr. Lorenz Lutherer, Ms. Sue Joanning, Mr. Lynn McMahon and Ms. Sue Willis for their help in making this thesis possible.
11
TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
LIST OF TABLES.......................................
v
LIST OF FIGURES......................................
v1
CHAPTER l
INTRODUCTION ............................. .
l
CHAPTER 2
LITERATURE REVIEW ........................ .
3
Regulation and Control of
CHAPTER 3
Fibrinolysis..............................
3
Structure of Plasminogen..................
4
Properties of Plasmin.....................
7
Immobilization Methods for Enzymes........
12
Kinetic Data Reduction....................
18
MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . ..
26
Soluble Assays............................
26
Immobilizing Procedure....................
28
Packed Bed Reactor Methods................
29
Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
Bed Packing Procedure.................
29
Data Procurement......................
31
-antiplasmin Assays....................
31
Fibrin Degradation Products Assay.........
32
a
2
111
PAGE Data Reduction............................
33
Soluble Assays........................
33
Insoluble Kinetic Parameters..........
33
RESULTS AND DISCUSSION....................
35
So 1 ub 1e P1 a smi n Stud i e s . . . . . . . . . . . . . . . . . . .
35
Immobilization of Plasmin.................
41
Immobilized Enzyme Reactor................
44
CONCLUSIONS AND RECOMMENDATIONS...........
55
Conclusions...............................
55
Recommendations...........................
55
LIST OF REFERENCES...................................
57
APPENDIX l
NOMENCLATURE.............................
61
APPENDIX 2
SOLUBLE DATA.............................
64
APPENDIX 3
PACKED-BED DATA..........................
69
CHAPTER 4
CHAPTER 5
lV
LIST OF TABLES PAGE Table 1 Kinetic Parameter Estimates for So 1ub 1e P1a smi n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
Table 2 Kinetic Parameter Estimates for Insoluble Plasmin............................
49
v
LI ST 0F FIGURE S PAGE Figure
1. Schematic of fibrinolytic system (showing the interaction of the components of the fibrinolytic system 1n vivo)...........................
5
2. Structure of plasminogen (the am1no acid sequence of human plasminogen).......
6
3. Schematic representation of the interaction between plasmin and a -antiplasmi n....................... .. . . 2 4. Structure of S-2251.. ....... ..............
10
5. Possible effects of immobilization on enzyme substrate reaction..............
15
Figure
6. Example of ideal Cornish-Bowden plot......
20
Figure
7. Packed-bed reactor setup..................
30
Figure Figure
Figure Figure
13
Figure 8. Plot of reaction rate vs. substrate concegtration for soluble enzyme at 25 C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~igure
9. Plot of reaction rate vs. substrate concentration for soluble enzyme at 37°C with and without a antiplasmin.. .. . .. .. .. .. .. 2.... .. .. . ... ... ..
38
39
Figure 10. Plot of reaction rate vs. substrate concegtration for soluble enzyme at 50 C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
Figure 11. Loss of activity for immobil3zed enzyme kept in solution at 4 C............
43
Fi gur e 12 . F0P 1eve 1 for con t ro 1 (x ) , i n sol ub 1e plasmin (e) and soluble plasmin (o)..... ..
45
Figure 13. Plot 8f packed-bed reactor data at 25 C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vl
47
PAGE
Figure 14. Plgt of packed-bed reactor data at 37
c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Figure 15. Plot of reaction rate vs. substrate concentration for packed-bed reactor at 37°C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
51
Figure 16. Plot of reaction rate vs. flaw rase for packed-bed reactor at 37
c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
52
Figure 17. Plot of log (V) vs. 1/T for packed-bed reactor at flow rate of 35 ml/hr and S
0
=
0 . 0 4 mM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vll
54
CHAPTER 1 INTRODUCTION Human blood contains an enzymatic system, known as the fibrinolytic system, which 1s responsible for dissolving blood clots. The formation of a clot in a blood vessel is usually in response to injury or due to disease. The clot is composed of a blood component, fibrin, which polymerizes and crosslinks to itself to form a network. A state of diminished blood flow is produced and this in turn initiates the process of dissolution, or fibrinolysis. Plasmin is the principal enzyme responsible for
breaking down fibrin polymers into fragments which are
soluble and can be carried
away
by the
blood stream.
In
response to the presence of fibrin, the vessel wall re 1eases tissue plasminogen activator,
an
enzyme which converts the
inactive plasminogen to its active form, plasmin. Also present in
the
called
bloodstream
is
a fast-acting
inhibitor of plasmin
-antiplasmin. Any excess plasmin produced or which is 2 released from the clot as it dissolves will be rapidly a
inhibited by
a -antiplasmin.
2 when a natura 1 ve s s e 1 mu s t be rep 1aced by an art i f i c i a 1
prosthetic vessel, the initiation for fibrinolysis in vivo is absent. blood
This is due to the absence of the cells which line vessels
and
release
tissue
1
plasminogen
activator
1n
2
response to fibrin clot formation. Therefore, vascular prostheses are much more likely to become occluded by blood clots. Currently, research is aimed at increasing the fibrinolytic activity of vascular prosthetic materials. Immobilization of plasminogen activators, plasminogen or plasmin, or inhibitors of fibrin formation may prevent clot formation for an extended period of time. With natural materials, endothelial cells will slowly grow 1n from both ends of the prosthesis and aid long-term fibrinolysis. This thesis investigated the kinetic properties of plasmin 1n the sol ub 1e and the immobilized state. Immobi 1 i zed plasmin was attached to powdered collagen, which is a natural material found extensively in the human body. The principal goal of this project
was
to
determine
if
the
reactive
selectivity
of
immobilized plasmin has been altered. Will it still react with fibrin, while its reaction with completely blocked?
a
-antiplasmin is partially or 2
CHAPTER 2 LITERATURE REVIEW Regulation and Control of Fibrinolysis In mammals blood clot formation occurs 1n response to a tear 1n the blood vessel, to stem the flow of blood. Clots may also form on the interior walls of vessels which are damaged or have developed sclerotic plaques. The principal effector for removal
of
coordinated
blood
clots
1s
the
actions of enzymes,
provides local
fibrinolytic activators,
system.
The
and inhibitors
reaction at the sites of fibrin accLJTiulation
without systemic effects.
The fibrinolytic system may be in
dynamic equilibrium with the coagulation system. Findings which show plasminogen activator activity in normal
blood support
this view (Sawyer, et al., 1960). The regulation and control of fibrinolysis appears to occur at several levels: release of plasminogen activator from the vascular wall, fibrin-associated activation of plasminogen by
and
inhibition of formed plasmin
a -antiplasmin. 2
The enzyme responsible for the breakdown of polymerized fibrin is called plasmin. concentration of 2.4
~M
It exists in the bloodstream at a
in the inactive form, plasminogen, and
may be converted to plasmin at the site of clot formation by plasminogen
activators
( Wi ll i ams, 3
et
a l. ,
198 3) .
The
4
plasminogen activators present in most tissues bind avidly to fibrin and their activity is greatly increased in the presence of
f i br i n
Plasminogen
( Cami l i o ,
et
al . ,
activators,
l 971 ;
urokinase
Ur a n o , and
et
al . ,
l 984 ) .
streptokinase,
are
commercially available and may be injected to enhance the level of fibrinolysis occurring in vivo. Plasmin may bind to fibrin 1 n the i n act i ve as we l l as t he act i ve state ( Al kj ae r s i g , e t al., 1959). Inactive plasminogen which is bound to fibrin can be converted to active plasmin and then the breakdown of the fibrin proceeds rapidly. The half-life of bound plasmin is two orders of magnitude longer than that of unbound plasmin. Any unbound
plasmin
inhibitor,
will
be
rapidly
inhibited
by the specific
a -antiplasmin (Mullertz and Clemmenson, 1976). The 2
breakdown of fibrin clots by plasmin can be followed by the measurement of fibrin degradation products appearing 1n the bloodstream. Figure l shows the fibrinolytic system in v1vo. Structure of Plasminogen Plasminogen molecular weight
is
a
single
of
about
chain
90,000.
glycoprotein
with
a
The complete amino acid
sequence has been determined and contains 790 ami no acids and is shown
1n Figure 2
(Collen, 1980).
Native
plasminogen has
NH -termi nal glutamic acid and can be easily converted to the 2 lysine form by limited plasmic digestion.
Lys-plasminogen is
~
F,..
Arlllk'umin
r ...~ • •
~
ed
:;:= ;;;;:: deV' prod!~ron
Figure 1. Schematic of fibrinolytic system (showing the interaction and actions of the components of the fibrinolytic system in vivo ) .
blood
IM .,.~-.:,,
(J1
6
Figure 2. Structure of plasminogen (the amino acid sequence of human plasminogen).
7
converted to p 1asmi n by the c 1eav age of a sing 1e Arg -V a 1 bond at position 560-561 two-chain
(Robbins, et al., 1967). This produces a
molecule composed
of a heavy chain,
or A chain,
coming from the NH -terminal part of plasminogen and a light 2 chain, or B chain, making up the C-terminal part. These two c h a i n s are connected by a s i ng 1e d i s u 1f i de br i dg e ( Wi man and Wallen, 1975). The heavy chain of plasmin contains five loop structures known as .. kringles, .. which contain the lysine binding sites involved in binding to fibrin. These sites are so named because they bind to lysine residues. This property is useful in the purification of plasminogen and plasmin using lysine-Sepharose (Deutsch and Mertz, 1970). The active site in the plasmin molecule is located in the light, or A, chain and is thought to involve the histidine-602, aspartic 1983).
acid-645,
serine-740
residues
(Williams,
et
al.,
The amino acid sequence of a peptide containing the
active site serine residue was determined by Grosskopf, et al., in 1969. Properties of Plasmin The main target of plasmin action, (Francis,
et
al.,
1980).
In
response
1n v1vo, 1s fibrin to fibrin formation,
plasminogen activator is released by endothelial cells. This
8
act i vat or has a hi gh af f i ni ty for f i br i n and for p1a s mi no gen attached to fibrin (Wiman and Wallen, 1977). The activation of plasminogen proceeds on the fibrin polymer surface, fibrin 1s lysed and activator and plasmin are released and bound by their respective inhibitors. Plasminogen may also be bound to fibrin as it polymerizes. Activators may then diffuse through the clot and
plasmin
may
exert
its
influence
relatively
free
of
inhibitors. Also the binding of plasmin to fibrin protects it from a 2-antiplasmin s1nce the lysine binding sites are involved 1n the binding to fibrin and therefore are not available for binding to antiplasmin (Collen, 1978). It has also been proposed that plasmin-inhibitor complexes formed in the
circulation may
{Ambrus and Markus,
dissociate in the presence of fibrin 1960).
This would provide a source of
plasmin immediately available to the site of fibrin formation. As already stated, plasmin can specifically bind to fibrin t hr ough i t s 1y s i ne bi nd i ng s i t e s {Wi ma n and Wa 11en , 19 77) . I t has been shown recently that although these sites are important 1n
the
binding
of
plasmin
to
fibrin,
the
active
site
interactions of plasmin with fibrin are at least as important in the activity of plasmin toward fibrin.
Morris,
{1981), found that a digested form of plasmin (Val
et al.
442 -plasmin) which lacked a major portion of the heavy chain and all of the lysine binding sites retained its ability to lyse fibrin.
9
Smith, et al. (1981), studied the fibrinolytic effects of an a cy 1at ed de r i vat i ve of p1as mi n. Th i s mo 1ec u1e ha s an ac y 1 group attached to the active site of plasmin. Therefore, it does not react with
a -antiplasmin or substrate, but it can
2
still bind to fibrin via the free lysine binding sites. Once bound it s 1owly becomes deacy 1ated and begins the breakdown of the fibrin clot.
By complexing the active site, plasmin 1n
solution may be protected from inactivation by a -antiplasmin 2 long enough to allow it to reach a fibrin clot where its action 1s needed. The reaction between plasmin and rapid
and Clemmenson,
-antiplasmin 1s very 2 1977; Wiman and Collen,
binds reversibly,
then forms a stable 1:1
(Christenson
1978).
It first
a
stoichiometric complex. As seen 1n Figure 3, a covalent bond forms between the serine-740 of plasmin and a leucine residue near the carboxy terminal end of fragment
-antiplasmin, releasing a 2 of MW 8000 from the inhibitor (Wiman and Collen, a
1979). Although the primary reaction is with the active center, the speed of binding 1s influenced by the availability of the lysine binding sites. If the lysine binding sites are complexed with s -aminocaproic acid, the inhibition of plasmin is slowed considerably
(Takada,
solution
inactivated
1s
et
already bound to fibrin.
al., much
1980). Similarly, faster
than
plasmin 1n
plasmin which 1s
Thus, fibrin inhibits the reaction
1
Ala
---
HO - Leu - Ph•----------------
V.1I - H
'---~~------~Arg - OH
Figure 3. Schematic representation of the interaction between plasmin and a -antiplasmin. 2
-------V•I-Gin - Gtu - Gin - A~>n - H
LBS
Lys -H
HO -Asn------------,
'--------------J ~-------------J
~
E
LBS II
2
pl~sm o n
anl op l .tsm on
B cha on o l
Ptg act
A Chllon ot C"smon
~
0
11
between plasmin and
a -antiplasmin through a lysine binding
2
site mediated interaction with a dissociation 6 7 10- -10- M (Wiman, et al., 1979). From the known concentrations of 1 wM)
and
plasminogen
(1.5
constant of
-antiplasmin (about 2 2.0 wM) in plasma and the
-
a
dissociation constant of 4 x 10- 6 Mit is concluded that about 30%
of
the a -antiplasmin 2
plasmin. Thus,
in circulation
is complexed
to
a -antiplasmin rapidly binds plasmin in plasma,
2 and thereby protects fibrinogen,
but as soon as p 1asmi n is
produced
1s
1n
excess,
fibrinogen
rapidly
degraded.
The
half-life of free plasmin is about 100 ms and the half-life of fibrin-bound
plasmin
is
about
s.
10
Efficient thrombolysis
therefore requires the adsorption of plasminogen activator and plasminogen to the fibrin surface and generation of plasmin out of reach of the fast-acting of
the
fibrinolytic
a -antiplasmin, or the activation
2 system to
such
an
extent
that a 2
antiplasmin is exhausted (Collen, 1980). Synthetic substrates for the determnation of plasmin were produced by mimicking a short sequence of peptides found at the plasmin
cleavage
Aurell,
1981).
site This
of
natural
peptide
substrates
sequence
is
then
(Claeson attached
and to
p-nitroanilide. When plasmin cleaves the peptide bond and p-NA is released, a yellow color is produced which can be followed spectrophotornetrically
at
405
nm.
The
most
widely
used
12
chromogenic
substrate
for
p1asmi n
1s
H-0-Va 1-L -Leucyl-L-
Lysine-p-nitroanilide (see Figure 4). The following reaction sequence is proposed by Friberger, et al. (1978): kl
E + S(
k2
> ES
-----J)
k_l
ES
l
I
E+
+ p
k3
s·
This sequence will g1ve the standard Michaelis-Menten equation for the reaction rate,
v=
Vmax S K + S m
for k3 >> k2 , and us1ng the steady state assumption that dES/dt i s appro x i rna t e 1y zero ( Fr i berger , 1982 ) . Thi s eq uat i on a11 ow s comparison of experimental findings with an idealized model and determination of the characteristic enzyme reaction parameters,
Immobilization Methods for Enzymes A specific conformation and an active center interacting with the substrate are regarded as essential features of the catalytic activity of enzymes.
The active center is usually
co mp o sed of sever a 1 ami no ac i d r e s i dues he 1d i n a spec i f i c spatial relationship. The three-dimensional conformation of the
13
ENZYME cleavage site
Synthetic substrate
Natural substrate
The synthetic substrate is made to mimic the natural substrate.
H3C
"- / H 3C CH 3 \ / CH 0
CH 3
NH 2 I CH 2 I CH 2
I
CH I CH 2 0
CH 2 I CH 2 0 I II 1 II I II~ 11 2 N- CH- C- HN- Cll- C-liN-CH- C -liN-~ -NO,
The structure of S-225 I, H-0- Val- Leu-Lys-pNA.
Figure 4. Structure of S-2251.
14
entire enzyme protein also has an important effect on the catalytic activity
activity. of
the
Consequently,
enzyme
1n
the
to
retain
the catalytic
immobilized state,
it
is
necessary to retain the native structure. If the amino acid residues at the active center, or the tertiary structure, are altered, the catalytic activity may decrease and changes of enzymatic properties such as substrate specificity may occur (see Figure 5). Functional
groups
suitable
for
enzyme
immobilization
include free amino and carboxyl groups, the sulfhydryl group of cysteine, the i mi dazo 1e group of his ti dine, pheno 1i c groups, and hydroxyl
group of serine and threonine. The reactions
leading to the immobilization of the enzyme should not involve functional groups in the active center. Methods for immobi 1 ization can be classified into three basic categories. 1)
Carrier-binding
method:
the
binding
of
enzymes to
water-insoluble carr1ers. 2) Crosslinking
method:
intermolecular
crosslinking
of
enzymes by means of bifunctional or multifunctional reagents. 3) Entrapping
method:
incorporating
enzymes
into
the
lattice of a semipermeable gel or enclosing the enzymes in a semipermeable polymer membrane.
15
EnzymQ
"\Substrato
7
~ Enzyme tn frtt aolutton
lmmoblltud Con form o t 1o ITO I
tnzy~
S ftnc tundra net
cho~t
Figure 5. Possible effects of immobilization on enzyme substrate reaction.
16
Physical entrapment techniques generally offer advantages of speed and ease of preparation over many chemical methods. The major difference between the entrapped and the chemic ally attached enzymes is that the former are isolated from large molecules which cannot diffuse into the matrix but the attached enzyme may be exposed to molecules of all sizes. Thus, for the assay of large substrates, such as proteins with proteolytic enzymes, an attached enzyme is required and not an entrapped enzyme (Bernfeld and Wan, 1963). Of the covalent binding techniques, the peptide binding methods
are
particularly useful
for
enzymes.
This
binding
method is based on the formation of peptide bonds between the enzyme and carrier. There are two major c 1asses of procedures available. 1)
Carriers containing carboxyl groups can be converted to reactive derivatives such as acyl
azide and reacted
with free amino groups in the enzyme. This method has been
employed
by
Coulet
and
Gautheron
(1976)
to
covalently attach enzymes to collagen films. 2) By using a condensing reagent for peptide synthesis such as c arbod i imide, peptide bonds are formed between free carboxyl or amino groups in the enzyme and amino or carboxyl groups 1n the carrier. The preparations are very
stable
and
a
number
of
successfully coupled by this method.
enzymes
have
been
17
Recent work in the area of immobilized enzymes has begun to concentrate more on the use of natural materials as carriers rather than synthetic resins (Kiraly and Nose, 1974). This is especially important for applications of immobilized enzymes in biological systems. Collagen is the rrost abundant protein 1n the human body. The fibrous nature of co 11 agen makes it very suitable for the immobilization of enzymes. Other factors which make it advantageous to use collagen are its high capacity for protein adsorption, its hydrophilicity and its ability to serve as a model of in vivo systems. Since most enzymes are globular proteins, side chains of polar amino acids are on the surface 1n aqueous solution. Therefore, hydrophilic carriers wi 11 tend to stabilize the enzyme. Collagen absorbs almost 500% (w/w) of water at pH 7.
At a pH of 3-4 collagen has a net positive
charge since amino residues will be protonated and rrost of the carboxyl residues will also retain their proton (Saini, et al., 1972).
Borchert and Bucholz controlling
the enzyme
(1983)
showed that by kinetically
adsorption
and
immobilization,
bio-
catalysts are produced which have much greater effectiveness under conditions of mass transfer limitation.
Provided that
there is strong adsorption, the protein is concentrated in the outer
shell
of
the
carr1er,
even
at
slow,
external
mass
transfer (Sh = 12). For Sh = 60, enhanced amounts of adsorbed
18
protein
are
obtai ned.
The
most
important
parameters
for
enhanced effectiveness are strong adsorption and short adsorption and coupling time. Several
components of the fibrinolytic system have been
successfully immobi 1 ized. Urokinase, a plasminogen activator, has been immobilized on nylon by means of covalent binding via reaction with carbodiimide (Sugitachi, et al., 1978). Urokinase has also been attached to collagen by means of crosslinking reaction with gl utera 1de hyde (Senatore, 1982). Streptokinase, another attached
plasminogen to
two
activator, polymers,
a
has
also
copolymer
been
covalently
of
p-amino-DL-
phenyl alanine and L-1 euci ne or a copolymer of ethylene and maleic anhydride,
and its action on plasminogen compared to
that of soluble streptokinase (Rimon and Rimon, 1974). They also immobilized plasminogen to these copolymers and then tried to activate the immobilized plasminogen with streptokinase, but were unable to detect any fibrinolytic activity. There was, however,
increased
proactivator activity of the immobilized
plasminogen in solution. Kinetic Data Reduction The
rates
of chemical
reactions
catalyzed
by
soluble
enzymes are dependent on substrate concentration, pH, temperature, solvent and presence of inhibitors. For most enzymes in
19
the absence of inhibitors, the reaction rate can be adequately described by the standard Michaelis-Menten equation:
v=
vmax s K + S m
Enzymes
obeying
this
rate
equation
are
said
to
follow
Michaelis-Menten kinetics. The parameters to be determined from experimental data using this equation are Km and Vmax· Km 1s a measure of the intrinsic reaction rates and is independent of enzyme concentration.
Vmax
is
directly proportional
to the
amount of enzyme (Bailey and Ollis, 1977). Estimates of Km and V max
may
be
determined
from
nonlinear
regress1on
of
the
experimental data or from a variety of linearized plots. The Cornish-Bowden
plot
(1978)
1s
a
particular
type
of
plot
obtai ned when the substrate concentration is plotted on the negative x-axi s
and
these points
are connected with their
respective reaction rate points plotted on the y-axis.
The
intersections of these lines each determines an estimate of Km and
V max
statistical
An example plot is shown in Figure 6. methods,
a
best
estimate
can
be
By us1ng
obtained
by
mi nimi zing the sum of the squares of the distances from this point to all the lines (Fischer, et al., unpublished report). For an enzyme immobilized on a solid support and studied 1n a packed bed reactor, the apparent kinetic parameters are
20
......, 0
,....-
D..
c
Q)
'U
3:
0
co I
....c::: V')
.,.... c
s.... u 0
,....ttj Q)
'U .,....
>< ttj
> >
40
E ,....V') I
Q)
,....-
D..
E
ttj
>
'-""
'
c
u
:J
,.......
•
r-
0
w
0
2
3
4
5
6
7
0.2 0.4
0.6
0.8
S(mM)
1
1.2
1
WITH '"'..l.ANTlPLASMIN
1.4
~ANTlPLASMIN
1.6
1.8
Figure 9. Plot of reaction rat~ vs. substrate concentration for soluble enzyme at 37 C with and without a -antiplasmin. 2
0
T
f
WITHOUT
2
~
w
E ....... >
0
~
-Ec
0
":J
•
r
"0w
0
,
2
3
4
5
6
7
8
9
10
.
. 0.4
.
t
. 0.8
.
~
S(mM)
1.2
1
+
I
I
, .6
I
I
2
2.4
I
Figure 10. Plot of reaction rate vs. substrate concentration for soluble enzyme at 50°C.
0
I
I +
~
0
41
decrease in reaction rate at high substrate or high product concentrations. Therefore, these influences were not considered in subsequent calculations. Addition of
a
2
-antiplasmin to the reaction mixture had a
significant effect on the reaction rate. V was decreased to max 7 3.3 x 10mol/min CU versus 7.08 x 10- 7 mol/min CU without
2 -antiplasmin. This decrease was not due to a decrease
a
in the reaction rate constant since K remained the same. The m
calculation of Vmax was based on the number of enzyme units added to the reaction initially. of
the
the
plasmin
number of enzyme
wi t h
the
substrate
calculating Vmax of
immediately
the
plasmin
-antiplasmin inhibits some 2 Therefore, and irreversibly. a
units actually available for reaction 1s
much
s mal l e r
t h an
t h at
us ed
1n
a -antiplasmin removed approximately half 2 added
initially
or
0.02
CU.
If
Vmax was
calculated based on the amount of uninhibited enzyme, the value would be the same as that obtained without inhibitor. Immobilization of Plasmin Using the procedure for immobilization presented 1n the previous section, 3-4
~g
of plasmin was immobilized to l mg of
collagen powder (10-40 mesh, dry). This amount gave a specific activity of 0.02-0.03 CU/mg collagen.
42
Stability of the immobilized preparation was determined both in the dry form and in solution. Tubes containing 0.5 mg of
collagen-plasmin
in l ml of
plasmin
diluent
were stored
at 4°C. The preparation was assayed periodically to assess loss of activity. Approximately 30% of the original activity leached out into the solution within the first 24 hours. At 48 hours the activity had dropped to 50% of the original. After this initial loss, probably due to leaching out of adsorbed but not bound plasmin, the activity remained constant for more than two weeks (see Figure ll). Due to these results, new preparations were .routinely left in solution for 24 hours prior to being dried and the activity determined. The dried preparation thus obtained retained its initial activity for more than 3 rronths at 4°C. To assess the effects of a -antiplasmin on the immobi2 lized plasmin, tubes containing approximately 0.5 mg of immobilized plasmin-collagen were reacted with S-2251 in the presence and absence of a -antiplasmin (1 :5 dilution of human 2 plasma). There was no difference in the reaction rate observed with
or
without
The specific activity 2 obtained with a -antiplasmin was 0.0267 + 0.002 CU/mg and 2 without a -anti plasmin 0.0256 + 0.005 CU/mg. Therefore, there 2 was no apparent i nhi bi ti on of the immobi 1i zed p1asmi n by a -antiplasmin. 2
a -antiplasmin.
~
0
~
"C 0
~
c
0
'-J
I)
t)
'... '
~ C)
...Q.
""'GCl.
0
10
20
30
-40
50
60
70
80
90
100
4
0 6
.
OCly~ c 4
8
. .
. (c) in so" Gly
10
.
,2
14
.
16
.
Figure 11. Loss of activity for immobilized enzyme kept in solution at 4°C.
2
. . . .
.
18
w
~
44
Another important test of the immobilized preparation was its effect on a synthetic clot. Clots were formed from thrombin and fibrinogen on ice in the presence of soluble or insoluble plasmin. The clot was then allowed to stand at 37°C for 3 hours and
at
room
temperature
for
supernatant were assayed for
24
hours.
Samples
of
the
levels of fibrin degradation
products (FOP). The soluble plasmin dissolved the clot within 3 ~g/ml.
hours with FOP level reaching more than 300 the
FOP
level
120
~g/ml.
At
for 24
the hours
insoluble the
plasmin
insoluble
was
enzyme
At 3 hours, more had
than almost
completely dissolved the clot with an FOP level of more than 300
~g/ml.
A control clot tube without plasmin was also run to
ensure that plasmin contamination of the reagents was not responsible for the lysis of the clots. The level of FOP in the control tube remained at approximately 8 period.
The
immobilized
plasmin
~g/ml
retained
for the 24 hour its
ability
to
di s sol ve a c l o t i n vi t r o . I n vi vo t e s t i ng of t he i mmo bi l i zed enzyme has not been done. FOP results are shown in Figure 12. Immobilized Enzyme Reactor Packed-bed reactor studies were conducted us1ng a column (1 em diameter) packed with 20 mg of immobilized enzyme mixed with 100 mg of regular collagen powder. This gave a bed height of 1.5 em. Substrate solution (0.03-0.30 mM) was pumped through
c
0
,...·r-
~
.......,
·r-
0
s::
0'1
.....>
•r-
s::
0'1
0..
0
·.c./)
.......,
·r-
>
QJ
s....
QJ
co
u
.......,
•r-
0
1
2
Time (hours)
3
24
Figure 12. FOP level for control (x), insoluble plasmin (e) and soluble plasmin (o).
1:32
1:64
~
U1
46
the reactor at flow rates varying from 35-120 ml /hr (Re 0.35-1 .4).
Product
concentration data
=
p steady state was
at
collected for each concentration and flow rate. XS
0
or product
concentration versus 1 n ( 1 - X) data were p1ot ted for each flow rate over the range of in 1et substrate concentrations (see Figures 13 and 14). These plots were 1i near for each flow rate (r = 0.96 - 0.99). K~ was determined from the slope of these 1 i nes and V•
max from the y-i ntercept. An average of the va 1ues obtained from each flow rate is reported in Table 2. The data obtained for the highest flow rate at 25°C was dropped and not included 1n the averages reported. Since the conversion for this temperature and flow rate was so low,< 0.1, the error in the data was significant. Also only three data points were available at this flow rate which made the calculated slope and intercept less accurate. At 37°C, K.. remained constant with increasing flow rate. m
This fact
indicated that
there was
no
observed
effect of
external mass transfer involved in this system. The presence of a significant amount of film diffusional effect should cause an increase in the apparent Km with a decrease in flow rate. The 1ow Re
p
i nd i c at ed t h at t here co u1d be a s i gn i f i cant
amount of external mass transfer influence. Using the analytical
procedure of
Patwardhan
and
Karanth
(1982),
the mass
transfer contribution was calculated and subtracted from the
47
O.D4
0.035 [
O.DJ
-D 0
+' L
+' (
•
0.025
0
(
0
u +'
0 (./')
X
O.D'l
0 J
"D 0
L
~
0.015
om
~18
~1~
~1
ln(l - X) ¢
78 mf'ri
A
114 m/rr"
Figure 13. Plot of packed-bed reactor data at 25°C.
48
0
O.ffi
(
-... 0
D
...(L
• 0
(
0
0
0 (,/)
0.{)3
X
...0
I
I
, J
0
L
a.
j
O.D2
I
I I
I I
O.D1
I
0
0 ~~----~~----~------~------------~ -{).1 0 -n2 -{).4 -0.3 ln(l - X) 0
76 rri/'rt
6
120 rri/tr
Figure 14. Plot of packed-bed reactor data at 37°c.
49
Table 2 Kinetic Parameter Estimates for Insoluble Plasmin 10 7 mol/min CU
K.. m
K• m
rrM
rrM
35
1 . 84
0.32
0. 21
54
2. 12
0.33
0.23
72
1 . 97
0.32
0.22
120
2.26
0.33
0.26
avg.
2.05
0.325
0.23
v·max
Flowrate ml/hr
37°C
+ so
-
+0. 18
-
X
+0.005
-
+0.02
-
35
1 . 10
0.292
0.25
48
1 . 08
0.259
0.22
76
1 . 17
0.290
0.25
avg.
1 . 12
0.28
0.245
25°C
+ so
-
+0.05
-
+0.02
-
+0.02
-
50
apparent Km. This procedure was followed for both 37°C and 25°C data. The results of this analysis showed that ksa increased with flow rate as expected. However since
K~
was constant for
varied flows, the results of adjusting the apparent Km for diffusional effects gave an intrinsic K which varies with flow m
rate. Since the intrinsic Km should vary only with temperature, this
result
was
not
consistent
with
esta~ished
enzyme
behavior. V'max for the i mmobi 1 i zed enzyme seemed to increase slightly with increasing flow rate. This increase can be due to better mixing which occurs at higher flow rates. The difference between the soluble Vmax and insoluble Vmax was also due to differences 1n mixing. Vmax will decrease significantly 1n an unmixed system, especially a two-phase system. Reaction rate versus substrate concentration was plotted for each flow rate (see Figure 15). This plot shows that the substrate concentrations emp 1oyed gave reaction rates sti 11 mostly in the first order reaction reg1on. Reaction
rate
versus
flow rate was plotted for each
substrate concentration (see Figure 16). A slight increase in reaction rate was seen with an increase in flow rate. The increase
was
due
to
a
decrease
1n
the
film
thickness
surrounding the enzyme particle with the higher flow rates, thereby decreasing the diffusional resistance.
51
0~ ~----------------------------------~
[
OM -
O.Dl-
0
I
o
I
o.D4
I
I
om
I
I
0.12
I
I
o.1s
I
I
01
I
I
01~
I
I
01B
StMute~
Figure 15. Plot of reaction rate vs. substrate concentration for packed-bed reactor at 37°C.
52
e~ ~----------------------------------~
OlH -
0
D
"
-E (
\
o.m -
+
0
t
E
+
E
v
.,
"0
\
,
om -
ll.
0
0 0
0
om4
0~--,--~.--~,--~r~··•t~11--11--11--11----11--1T--1
0
~
~
m
m
100
1~
FbmJte (ml/tr)
Figure 16. Plot of reaction rate vs flow rate for packed-bed reactor at 37 6C.
53
An enzyme column was reacted with 0.04 mM substrate at the 1owest 42 °c,
flow rate.
The terrperature was
varied
from 4°C to
an d out 1et product concentration at each terrperature
collected at steady state. A plot of log (v) versus 1/T showed two
distinct
controlled
linear
regions
region from
(see Figure
4°C to
25°C was
17).
A reaction
seen with
a slope
of -Ea/R = -2700 which gives Ea = 5.35 kcal/mole .. Another region from 32°C to 42°C gave a slope of -800 and Ea
=
1.58
kcal/mole. For reactions in the strong pore resistance regime, the effectiveness factor, n , is approximately equal to 1/¢ , where
()"1
CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS Conclusions The results of this project have shown that plasmin can be cov a 1ent 1y i mmobi 1i zed onto co 11 agen. This immobi 1i zed plasmin retained its fibrinolytic activity,
its ability to dissolve
synthetic clots. The immobilized plasmin was not susceptible to inhibition by a -antiplasmin as was the soluble enzyme. These 2 facts rna k e t he pos s i bi l i ty of i t s use i n vas c ul ar prostheses more plausible. The apparent K for the immobilized enzyme was the same as m
that obtai ned for the soluble enzyme at both 25°C and 37°C. There was a 1so no variation in the apparent Km with varying flow rate. The conclusion is that this Km is actually the intrinsic K for the immobilized enzyme. The maximum reaction m rate was decreased for the insoluble enzyme from that obtained for the soluble enzyme. A change in the Km may indicate some conformational change due to the immobilization or increased steric hindrance for the substrate. This was not evident. Recommendations l) Optimize immobilization conditions to obtain maximum enzyme activity. Test different pH and enzyme levels for increased 55
56
enzyme adsorption. Vary coupling and/or adsorption times to see if increased activity is obtained. 2) Obtain pH-activity profile of immobilized enzyme. 3) Obtain estimates of Km and Vmax using well-mixed system. 4) Attempt to ascertain the specific site of the covalent attachment. 5) Test immobilized enzyme 1n an 1n vivo system.
LIST OF REFERENCES Alkjaersig, N., A. P. Fletcher and Sherry, S., "The Mechanism of Clot Dissolution by Plasmin," J. Clin. Invest., 38:1086-1095 (1959). Ambrus, C. M. and Markus G., "Plasmin-Antiplasmin Complex as a Reservoir of Fibrinolytic Enzyme," Amer. J. Physiol., 199:491-494 (1960). Bailey, J. E. and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw Hill Book Co., New York, NY (1977). Bernfeld, P. and Wan, J., "Antigens and Enzymes Made Insoluble by Entrapping Them Into Lattices of Synthetic Polymers," Science, 142:678-683 (1963). Borchert, A. and Buchholz, K., "Improved Biocatalyst Effectiveness by Controlled Immobilization of Enzymes," Biotechnol. Bioeng., 26:727-736 (1984). Brownlee, M., Vlassara, H. and Cerami, A., "Nonenzymatic Glycosylation Reduces the Susceptibility of Fibrin to Degradation by Plasmin," Diabetes, 32:680-684 (July, 1983). Camilio, S. M., Thorsen, S. and Astrup, T., "Fibrinogenolysis and Fibrinolysis with Tissue Plasminogen Activator, Urokinase, Streptokinase-Activated Human Gl obu 1 in and Plasmin, .. Proc. Soc. Exper. Biol. Med., 138:277-280 (1971). Chi bata, S. , Ed. , Immobi 1 i zed Enzymes, Research and Deve 1opment, Halsted Press, New York, NY (1978). Christensen, U., "Requirements for Valid Assays of Clotting Enzymes Using Chromogenic Substrates," Thrombos. Haemostas, 43:169-174 (1980). Christensen, U. and Clemmensen, I., "Kinetic Properties of the Primary Inhibitor of Plasmin from Human Plasma," Biochem. J., 163:389-391 (1977).
--
Claeson, G. and Aurell, L., .. Small Synthetic Peptides with Affinity for Proteases in Coagulation and Fibrinolysis: An Overview," Ann. N.Y. Acad. Sci., 370:798-811 (1981).
57
58
Collen, D., "On the Regulation and Control of Fibrinolysis," Thrombos. Haemostas, 43:77-89 (1980). Cornish-Bowden, A., Fundamentals of Enzyme Kinetics, Butterworths, London (1979). Coulet, P. R. and Gautheron, D. C., "Enzyme Anchoring on Chemically Activated Collagen Membranes," in Analysis and Control of Immobilized Enzyme Systems, D. Thomas and J. P. Rernevez, Eds., North Rolland, New York (1976). Deutsch, D. G. and Mertz, E. T., "Plasminogen: Purification from Human Plasma by Affinity Chromatography," Science, 170:1095-1096 (1970). Fischer, P. E., Senatore, F. F. and Venkataramani, E. S., "Explicit Expressions for Michaelis-Menten Kinetic Parameters from the Direct Linear Plot," submitted to Biochemistry (1985). Francis, C. W., Narder, V. J. and Barlow, G. H., "Plasmic Degradation of Crosslinked Fibrin," J. Clin. Invest., 66:1033-1043 (1980). ------Friberger, P., Know, M., Gustavsson, S., Aurell, L. and Claeson, G., "Methods for Determination of Plasmin Antiplasmin and Plasminogen by Means of Substrate S-2551," Thrombos. Haemostas, 7:138-145 (1978). Groskopf, W. R., Summaria, L. and Robbins, K. C., "Studies on the Active Center of Human Plasmin," J. Biol. Chern., 244(13):3590-3597 (1969). Johnson, A. J., Kline, D. L. and Alkjaersig, N., "Assay Methods and Standard Preparations for Plasmin, Plasminogen and Urokinase in Purified Systems, 1967-1968, .. Thromb. Diath. Haemorrh., 21:259-272 (1969). Kiraly, R. J. and Nose, L., "Natural Tissue as a Biomaterial," Biomat., Med. Dev., Art. Org., ~(3):207-224 (1974). Kobayashi , T. and Moo- Young , M. , " Th e Ki n e t i c s and Ma s s Transfer Behavior of Immobilized Invertase on Ion-Exchange Resin Beads," Biotechnol. Bioeng., ]2:47-67 (1973). Kobayashi, T. and Moo-Young, M., "Backmixi ng and Mass Transfer in the Design of Immobilized Enzyme Reactors," Biotechnol. Bi oeng. , 13:89 3-91 0 ( 19 71 ) .
59
Lilly, M. D., Hornby, W. E. and Crook, E. M., 11 The Kinetics of Carboxymethyl-Cellulose-Ficin in Packed Beds, .. Biochemistry, 100:718-723 (1966). Marder, V. J., Cruz, G. 0. and Schumer, B. R., .. Evaluation of a New Antifibrinogen Coated Latex Particle Agglutination Test in the Measurement of Serum Fibrin Degradation Products, .. Thrombos. Haemostas, 37:183-191 ( 1977). Morris, J. P., Blatt, S., Powell, J. R., Strickland, K. K. and Castellino, F. J., "Role of Lysine Binding Regions in the Kinetic Properties of Human Plasmin," Biochemistry, 20 ( 17 ) : 4811 - 481 6 ( 1981 ) . Mu 11 ertz, S. Plasmin (1976).
and C1emmen sen, I. , 1n Human Plasma,"
The Primary Inhibitor of Biochemistry, 159:545-553
11
Patwardhan, V. S. and Karanth, N. G., "Film Diffusional Influences on the Kinetic Parameters in Packed-Bed Immobilized Enzyme Reactors, •• Bi otechno 1 . Bioeng., 24:763-780 (1982). Rimon, A. and Rimon, S., "Immobi 1 ized Components of the Plasmin System," in Insolubilized Enzymes, M. Salmona, C. Saronio and S. Garattini, Eds., Raven Press, New York, NY (1974). Robbins, K. C., Summaria, L., Hsieh, B. and Shah, R. J., "The Peptide Chains of Human Plasmin. Mechanism of Activation of Human Plasminogen to Plasmin,•• J. Biol. Chern., 242:2333-2342 (1967). Rovito, B. J. and Kittrell, J. R., ••Film and Pore Diffusion Studies with Immobilized Glucose Oxidase," Biotechnol. Bioeng., _]2:143-161 (1973). Sawyer, W. D., Fletcher, A. P., Alkjaersig, N. and Sherry, S., "Studies on the Thrombolytic Activity of Human Plasma," J. Clin. Invest., 39:426-434 (1960). Senatore, F. F., "Development of a Biocompatible Vascular Prosthesis," Ph.D. Dissertation, Rutgers University, Pascataway, NJ (1983). Smith, R. A. G., Dupe, R. J., English, P. D. and Green, J., ••Fibrinolysis with Acyl-Enzymes: A New Approxach to Thrombolytic Therapy, •• Nature, 290:505-508 (1981).
60
Sugitachi, A., Takagi, K., Inaska, S. and Kosaki, G., .. Immobilization of Plasminogen Activator, Urokinase, on Nylon, .. Thrombos. Haemostas, 39:426-436 (1978). Takada, A., Ito, T. and Takada, Y., .. Interaction of Plasmin with Transexamic Acid and a 2 -Plasmin Inhibitor in the Plasma and Clot, .. Thrombos. Haemostas, 43:20-23 (1980). Urano, T., Takada, Y. and Takada, A., The Enhanced Activation of Glu-Plasminogen by Urokinase in the Presence of Fibrin or DES. A Fibrin as Measured by the Release of B Peptide and FOP, .. Thromb. Res., 36:429-435 (1984). 11
Williams, W. J., Beutler, E., Erslav, A. J. and Lichtman, M. A., Hematology, 3rd Edition, McGraw Hi 11 Book Co., New York, NY (1983}. Wiman, B. and Collen, D., 0n the Mechanism of the Reaction Between Human a 2 -Antiplasmin and Plasmin, .. J. Biol. Chern., 254(18):9291-9297 (1979). 11
Wiman, B. and Collen, D., .. On the Kinetics of the Reaction Between Human Antiplasmin and Plasmin, .. Eur. J. Biochem., 84:573-578 (1978). Wiman, B. and Wallen, P., The Specific Interaction Between Plasminogen and Fibrin, .. Thromb. Res., ~:213-222 (1977). 11
Wiman, B. and Wallen, P., 0n the Primary Structure of Human Plasminogen and Plasmin: Purification and Characterization of Cyanogen-Bromide Fragments, .. Eur. J. Biochem., 57:387394 (1975). 11
Wiman, B., Lijnen, H. R. and Collen, D., "On the Specific Interaction Between the Lysine-Binding Sites in Plasmin and Complimentary Sites in a 2 -Antiplasmin and in Fibrinogen, .. Biochem. and Biophys. Acta., 579:142-154 (1979). Wiman, B. and Collen, D., .. Molecular Mechanism of Physiological FibrinolysiS, Nature, 272:549-550 (1978). 11
APPENDIX l NOMENCLATURE
61
62
NOMENCLATURE a
interfacial area per unit volume, cm-l
cu
casein unit, amount of enzyme which re 1eases 0.1
~mol
of tyrosine from a-casein per minute
particle diameter, m radial and axial dispersion coefficients, respectively diffusivity, cm 2/min flow rate through packed bed, cm 3/min intercept on ln(l -X), XS 0 axes, respectively Michaelis-Menten constant, (k_ 1 + k2 )/k 1 , mM intrinsic Michaelis-Menten constant for immobilized enzyrre apparent Michael i s-Menten constant for immobi1i zed enzyrre substrate film mass-transfer coefficient, em/min length of packed bed nkat
unit of enzyme which converts 1 x 10
-9
moles of
substrate per second under standard conditions 00
optical density particle Reynolds number, (dp
up/~
)
substrate concentrations in bulk and at catalyst surface, respectively
63
substrate concentration at reactor inlet and outlet, respectively S-2251
substrate, H-0-Val-L-Leu-L-Lys-p-nitroanilide
Sc
Schmidt number,
Sh T
Sherwood number, k d /0 s p · L/U m1· n-l space t 1me,
u
superficial velocity, em/min
ll/P
0
max1mum reaction rate for soluble enzyme max1mum reaction rate for immobilized enzyme 1n packed bed reactor X
conversion, (S 0
-
Se)/S 0
APPENDIX 2 SOLUBLE DATA
64
65
Reaction Rate Data Soluble Enzyme 25°C
OBS
S(mM)
1 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0.2266 0.45 33 0. 6798 0.9066 0.0504 0.0504 0.0504 0.1007 0.1007 0.1007 0.1007 0.2014 0.2014 0.2014 0.2014 0. 3021 0.3021 0. 3021 0.3526 0. 35 26 0.3526 0.3526 0. 4533 0.45 33 0. 4533 0.4533
V(mol/min CU) 1. 9825 3. 11 00 3.7310 4.25 65 1. 0408 0.7130 0. 7887 1 . 35 94 1. 3230 1 . 3289 1. 4160 1 . 94 20 2.2520 2.0514 2.0514 2. 83 60 2.3960 2.4030 2.7970 2.8360 2.3960 3.0405 3.6260 3.6390 2.9160 3.4110
66
Reaction Rate Data Soluble Enzyme 37°C OBS
S(mM)
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
0.0755 0.0755 0.0755 0.0755 0. 1888 0. 1888 0. 1888 0.3022 0.3022 0.3022 0.3022 0. 1888 0.4533 0.4533 0.4533 0. 67 99 0.6799 0.6799 0.6799 0.3022 0.3022 0.3022 0. 6044 0.6044 0.9066 0.9066 1. 2088 1 . 2088 1. 2088 1.5110 1.5110 1.5110
V(mol/min CU) 1.3197 1 . 12 20 1. 5560 1 . 21 30 2. 9560 3. 12 50 2.2300 3.8120 3.2120 3.2130 3. 0400 2. 19 90 3.7320 3.7330 3.8720 5. 23 50 4.6680 5.2580 5. 1180 2.4100 3. 187 0 3.5300 3.6930 5.4880 4.5870 5. 87 00 4.8780 6.4500 6. 5300 5.2080 6. 8400 6.6860
67
Reaction Rate Data Soluble Enzyme 37°C w/a -antip1asmin 2 OBS
S(mM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
0.1511 0.1511 0.1511 0.1511 0.3022 0.3022 0.3022 0.3022 0.3022 0.3022 0.3777 0. 37 77 0.3777 0.3777 0.4533 0.4533 0.4533 0. 5288 0.5288 0. 5288 0.5288 0.6044 0.6044 0. 6044 0. 9066 Oo9066 Oo9066 1 . 2088 1. 2088 1 02088 105110 1.5110 1.5110
V(mo1/min CU) l. 1460
0.9130 0.8552 1 . 0880 0.6413 1 . 09 20 0. 9 912 1.4380 l. 6330 1 .8850 l. 61 00 1.6520 1.82 70 2.0410 l. 7 760 2.3320 2.3130 1. 8850 l. 9046 2.0410 2.4170 2.0410 2.3130 1 . 9600 3.1090 2. 66 30 20 3320 3. 1560 3 01680 207210 3o6930 305560 208440
68
Reaction Rate Data Soluble Enzyme 50°C OBS
S(mM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
0.0755 0.0755 0.0755 0.1511 0.1511 0.1511 0.2266 0.3022 0.3022 0.3022 0.3022 0.3022 0.4533 0.4533 0.4533 0.6044 0.6044 0. 6044 0.6044 0.6044 0. 6799 0.6799 0.9066 1 . 2088 1. 2088 1 . 2088 1. 8132 1 . 8132 1.8132 1 . 8132 2.2540 2. 2540 2.2540 2. 41 76 2.4176 2. 41 76 2.7198 4.0797
V(mol/min CU) l. 017
l . 017 1.149 2.011 1. 998 2.197 4.266 3.541 3.635 3.787 2. 920 3.605 5. 331 5.4 70 6. 946 6.257 7. 139 5.330 3.580 4.398 7.057 7.230 7. 211 7.320 5.397 7.450 8.070 6.320 8.840 11 . 7 40 8. 970 7.000 9.060 9.818 8.526 10.930 13.390 13.5 50
APPEND IX 3 PACKED-BED DATA
69
70
Packed-Bed Reactor Data
F1owrate m1/hr 35
50
74
114
X
1n(1 - X)
0.0302
0.0070
0.23
-0.267
0.0604
0.0123
0.20
-0.2 26
0.1511
0.0260
0.17
-0. 190
0.3022
0.0415
0. 14
-0.148
0.0302
0.0056
0. 18
-0.208
0.0604
0.0093
0.15
-0.167
0.1511
0.0199
0. 13
-0. 141
0.3022
0.0310
0. 10
-0. 109
0.0302
0.0039
0. 13
-0. 141
0.0604
0.0066
0.11
-0.116
0.1511
0.0146
0.10
-0.101
0.3022
0.0220
0.07
-0.076
0.0604
0.0045
0. 07
-0.0772
0.1511
0.0100
0.06
-0.0685
0.3022
0.0151
0.05
-0.0513
71
Packed-Bed Data Mass Transfer Analysis ( 2 5°C)
F-0.493 5.73
1.037
Plot
4.75
0.869
yi e 1ds
5.304
0.727
m1n. and 1/>-
5.176
0.579
1 . 27
of T/1
vs.
1 y-i ntercept
F- 0 · 493 = 4. 6
= 0.784 =
>-
ksa = >-(F0.493)
F1owrate m1/hr
ks a
K" m
K• m
35
1 . 2 25
0.296
0.254
50
1. 46
0.259
0.224
76
1 . 75
0.290
0.258
114
2.19
0.390
0.357
=
72
Packed-Bed Reactor Data (37°C) F1owrate m1/hr 38
54
72
120
s0
xs 0
mM
JTtv1
0.0302
0.0094
0.31
-0.37 4
0.0504
0.0152
0.30
-0.3 59
0.0755
0.0206
0.27
-0.319
0.1511
0.0377
0.25
-0.287
0.3022
0.0586
0.19
-0.216
0.0302
0.007 45
0.25
-0.283
0.0504
0.01235
0.24
-0. 281
0.0755
0.0166
0.22
-0.248
0.1511
0.0309
0.20
-0.229
0.3022
0.0469
0. 16
-0.168
0.0302
0.0056
0. 18
-0.203
0.0504
0. 0092
0. 18
-0.201
0.0755
0.0124
0.16
-0.180
0.1511
0.0232
0. 15
-0.166
0.3022
0.0336
0. 11
-0. 117
0.0302
0.0038
0. 13
-0. 137
0.0504
0.0064
0.12
-0. 136
0.0755
0.0087
0. ll
-0. 123
0.1511
0.0160
0. 10
-0. 112
0.3022
0. 0240
0. 08
-0.081
X
1n ( 1 - X)
73
Packed Bed Data Mass Transfer Analysis (37°C) F-0.493 3.74
l. 008
Plot
vs.
F- 0 · 493
3.35
0.837
yields y-intercept
= 2.44
3.47
0.727
min. and l I A = l . 252 = A =
3. 12
0.565
0. 799
ksa = A(F0.493)
Flowrate ml/hr
of T/ll
m = Km 1
K
11
-
max /k s a
V
ks a
K..
K•
38
0. 792
0.323
0.214
54
0.954
0.335
0. 231
72
l .099
0.320
0.223
120
1. 414
0.329
0.261
m
m
