AIR-Reoxidation of reduced, denatured chymotrypsinogen A covalently attached to a solid matrix

Vol. 48, No. 5, 1972

BIOCHEMICAL

AIR-REOXIDATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF REDUCED, DENATURED CRYMOTRYPSINOGEN A

COVALENTLY ATTACHED TO A SOLID MATRIX' John C. Brown,

North Received

Harold

E. Swaisgood,

and H. Robert

Departments of Biochemistry Carolina State University,

July

Horton

and Food Science Raleigh, N. C. 27607

14,1972

SUMMARY: Bovine chymotrypsinogen A was covalently attached to porous succinylglass beads via the zymogen's amino groups. The bound zymogen was completely reduced in 8 M urea, then allowed to reoxidize, and activated to chymotrypsin. Comparison of the ko (catalytic coefficient) of this preparation with that of a similar preparation which had never been exposed to reductant showed a 53 % recovery of esterolytic activity towards N-benzoyl-L-tyrosine ethyl ester. Values of Km and k, for non-reduced, matrix-bound zymogen, following its activation to chymotrypsin, were 12.8 x 10-5 M and 11.0 sec'l, respectively. Corresponding values for reduced, air-reoxidized preparations were 6.8 x 10m5 M and 3.1 set-l. Attempts state

to reoxidize

following

the zymogen bility

the

chymotrypsinogen

complete

have met with

of the reduced

esterolytic

activity

was only

1.4 % (2).

as to whether

the

structure

in zymogens

(such

sequence,

it

of the thus

through ovalbumin),

achievement

five

array

bonds

to the

acids

to circumvent

determined

the

tertiary by the

comprising problems

addition

investigate

functional

is

(1,2).

of potential

to further

of biologically

in

insolu-

of pH and the

the maximum recovery In order

present

relative

to air-oxidation

manipulation

of amino

became necessary

functional

disulfide

amenable

as chymotrypsinogen)

linear

biologically

due in part

conditions

(e.g.,

question

thermodynamics

under

was attained

separators

of the

success,

protein

Even when solubility of molecular

reduction little

A to its

inherent

the primary of aggregation

and

insolubility. Recently, muscle

lactate

Cho and Swaisgood dehydrogenase,

exposure

to 7 M guanidinium

nificant

recoveries

(3)

demonstrated

covalently

bound

chloride.

of enzymatic

Epstein

activity

the

reactivation

to porous and Anfinsen

glass (1)

of rabbit beads,

sig-

when carboxymethylcellulose-bound

trypsin was subjected to reduction and air-reoxidation. cu-Chymotrypsin found to be highly active after immobilization by attachment to porous beads

following

observed

was glass

(4).

lThis investigation has been supported in Research Grant GB-78949, and in part by medical Sciences Support Grant RR 07071, Schools of Agriculture and Life Sciences ences. Paper3820 of the Journal Series Agricultural Experiment Station, Raleigh, Copyright 0 1972 by Academic Press, Inc. AN rights of reproduction in any form reserved.

1068

part by National Science Foundation National Institutes of Health Bioand is a contribution from the and Physical and Mathematical Sciof the N. C. State University N. C.

Vol. 48, No. 5, 1972

In view

of these

chymotrypsinogen covalent

BIOCHEMICAL

findings,

which

attachment

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

we sought

had undergone

to porous

reductive

glass

chased

from

Bovine Pentex

ton Biochemical Gdn.HCl

Pierce

Chemical

Porous

alkylaminosilane-glass

obtained

from Corning

Preparation H20,

4.5, for

Coleman

and degassed.

of the

The solution

of the remaining

beads,

corresponding

pH 7.0,

After

for

and then

H20.

Effluent

distilled

522 a pore

from

grade J. T. Baker

diameter)

recycling,

was essentiallv with

were

that

After

of solid

with

disat pH recycling

to 0.2 M in EDC by for

5 hr.

To activate

the

EDC were

were

acid

continuous

washed

A quantity succinyl

made to the

30 min of recycling

the beads

of Cho

1 M NaCl and then

was continued

13.5 mg of CTGn was added

treated

experiments.

with

Company;

from ethanol.

control.

two additions

24 hr at O".

1 M NaCl-0.08

eluted

from Worthing-

after

with

each

0.1 M sodium

at 0 O.

with

Analyzer.

urea

was increased

for

to 0.1 M),

final

to all

Amino Acid -Acid

the

washing,

was recycled

grade

reagent.

solution

and recycling

phosphate,

prior

the

removed

After

trypsin

Sigma Chemical

was made 0.5 M in succinic

reagent,

(each

was pur-

EDC and sequanal

mesh,

of solid

was then

addition,

washed

(40-60

solid

solution

buffer

and Bell;

beads

of succinylated

its

Works.

12 hr at room temperature,

groups

bovine

by recrystallization

beads Glass

of

crystallized,

from

and reagent

purified

and 0.1 M in EDC by addition

addition

times

of SG-CTGn Beads. The procedure --(3). A column of beads was washed

and Swaisgood tilled

A, six

Company;

and further

Company,

following

BTEE* was obtained

from Matheson,

from

Chemical

denaturation

twice-crystallized

Corporation.

B-mercaptoethanol

air-reoxidation

AND METHODS

chymotrypsinogen

Corporation;

the

beads.

MATERIALS Materials.

to examine

with

Analysis.

The control CTGn.

SG-CTGn beads, was collected

reaction, buffer,

were

were

performed

followed

in dialysis to a Pyrex

pH 7.8.

tubing,

washed

types

solution in phosphate

of beads

Beads were

on a Beckman Model 0.284

by several

vial,

and the

also

both

1.2 ml representing

10 ml of 6 M Gdn-HCl, H20, transferred

After

M Tris-chloride

Analyses

to the beads,

beads

washings

dialyzed

and taken

g dry weight, with

116 Amino were against

The beads

*The abbreviations used are: BTEE, N-benzoyl-L-tyrosine ethyl ester; DTNB, 5,5'-dithio-bis(2-nitro1-ethyl-3-(dimethylaminopropyl)-carbodiimide; benzoic acid); BME, @-mercaptoethanol; GdneHCl, guanidinium chloride; bovine chymotrypsinogen A; SG-CTGn, succinyl-glass-chymotrypsinogen; succinyl-glass-chymotrypsin; and TNB, thiolnitrobenzoate.

1069

degassed

distilled

exhaustively

to dryness.

were

EDC, CTGn, SG-CHT,

Vol. 48, No. 5, 1972

were

dried

BIOCHEMICAL

and weighed,

in evacuated

vials

and both

placed

content

of each was calculated present

unless

beads

otherwise

to determine

observed

interbead

volume

(V,)

culation

hydrolyzed

for

of average not

occupied

in

24 hr.

of Asp,

to Gdn.HCl

identically

by a column

6 N HCl

The protein

recoveries

exposed

(Vm) of a weighed

of 2.18

pore

g/cc

(6).

(VP).

were

Gly, also

each time,

of wet beads

quantity

was used

Total

of dry beads

volume

(Vt)

Vt - Vm = V, + VP

by transferring

and measuring

the beads

the volume

= V,,

to a syringe thereby

occupied,

=

Vm +

the void

filled

permitting

with the

cal-

of V,.

Activation

of SG-CTGn to SG-CHT. A solution of trypsin in 0.08 M Tris-Cl---pH 7.8, was recycled through the beads for 2 hr at room temperature.

0.1 M CaC12,

Denaturation, denaturation However, dure

+

Vt was determined

H20, degassing,

bath

to pack almost

the volume

volume

space

were

dry weight.

Void Volume. The matrix -was determined using a density volume.

toluene

SG-CTGn beads

were

stated,

their

and beads

on the basis

in CTGn (5).

Since

effluent

in a refluxing

and Ala analyzed.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Reduction,

and Reoxidation. was essentially that

in 8 M urea sulfhydryl

(7),

groups

in that

the

TNB anion

10 % BME in 0.5 M sodium Reoxidation beads

for

Assay.

was pumped through

was monitored

and Anfinsen

(1).

of Ellman's

glass-bound

phosphate

reductive

protein

buffer,

0.05 M Tris-Cl,

procewith

pH 7.5.

pH 8.6,

through

the

Cp is

columns

at 3 ml/min,

activated

activated, and the

of the Michaelis-Menten

=

the

- f)

Km.ln(l

concentration;

to product; E,,

similarly

equation

enzyme was utilized

the product

enzyme;

0.1 ml of SG-CTGn beads,

were

to SG-CHT, used.

absorbance

BTEE

of the

at 256 nm (8). form

of surface-bound

been converted of the

from the

sodium

SG-CTGn beads,

cP where

was eluted by recycling

For assay, the bead

The integrated a column

of Epstein

by a modification

acetate-O.OlM

was effected

ml or reoxidized

effluent

titrated

for

24 hr at room temperature.

Kinetic and 0.35

were

The procedure

k,, total

f,

the

overall

enzyme

in evaluating

for

the kinetics

of

the data:

+ k,E,V,/Q the

fraction

of substrate

rate

constant

concentration;

V,,

Q, the

(catalytic the void

which

has

COeffiCieIIt)

volume;

and

flow rate. This equation differs slightly in that we have used the void volume rather than

from that of Lilly --et al. the total column volume in

determining

analyzed

least

enzyme concentration.

The data

squares.

1070

were

by the method

of

(9)

Vol. 48, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Characteristics

of Non-Reduced Activated

and Reduced-Reoxidized to SG-CHT

Total Volume (ml>

Dry Weight 63)

Native

0.10

0.028

0.086

3.26

Reoxidized

0.35

0.093

0.301

1.16

Matrix-bound Enzyme

V~~~~,

SH Content (Reduced) (moles/mole)

K, CM)

x 1O-3

-

12.8 x 1O-5

11.0

x 10-2

11

6.8 x 10-5

3.1

Protein (mg)

(ml)

SG-CTGn

ko (set- 1)

RESULTS Table "native"

I gives

the characteristics

referring

not been

reduced,

8 M urea

and allowed

and "reoxidized"

0.14 mg protein/g mately tion

the

old

its

dry beads.

same value;

Analysis

hence,

there

to that

protein

appeared

beads,

even without

exposure

Accordingly,

these

to GdnsHCl, stored

eluate

the beads as amino

acid

were

approxi50 % adsorp-

stored

in cold

analysis of 0.14

used

for

in

revealed

yielded

were

gave a value

preparations

had

had been reduced

of the beads

to have been at least if

was released,

dry beads.

SG-CHT) which

which

analysis

of the Gdn-HCl

However,

of SG-CTGn columns,

product,

Amino acid

to the beads.

H20, adsorbed

activation

referring

to reoxidize.

of the zymogen

distilled

of the two types

to SG-CTGn (and

of 30-day mg protein/g

enzymatic

studies. Non-reduced 10-4 M and a

of 11.0 . . upon activation,

SG-CTGn, tively

SG-CTGn, k,

(Table

I).

of esterolytic following data

fitted

tude

of errors

changes if

the

to the

integrated

encountered

in flow flow

to enzyme.

rate

rate

rate

substrate

can result

is < 1 ml/min,

inhibition,

same value of Cp as did 0.27 u&i. Control SG-CHT beads exhibited ranging

from

ko/Km

yielded

of 3 mljmin, x lOa

indicates

by air-reoxidation Figure equation,

in sizeable

a l$.,, of whereas

1.28 x

reoxidized

M and 3.1 set-l,

ratios

It

reduced

a typical

and serves

error.

zymogen,

plot

to indicate

was found

experimental

respec-

a 53 % recovery

of the

1 presents

in such measurements.

the data plot in an almost vertical more than 25-30 % of the substrate exhibited

rate

of 6.79

of the

is achieved

activation

to SG-CHT,

at a flow

g ave values

Comparison

activity its

upon activation set"

of the the magni-

that

slight

In our

system,

too much of the substrate is hydrolyzed so that Best results are obtained when not line. has been hydrolyzed. in that

a concentration

no esterolytic

0.5 to 3 ml/min.

1071

The preparations of 0.54 mM produced

activity

at flow

rates

also the

Vol. 48, No. 5, 1972

BIOCHEMICAL

-32 -26-24

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-20 -16 -12 -6 In(l-f

ln(l - f) vs. and Reoxidized

Fig. 1. A Plot of Cp SG-CHT (open circles),circles).

-4

)x 102

for Non-Reduced SG-CTGn Activated to SG-CTGn Activated to SG-CRT (filled

DISCUSSION To the authors' zymogen with exist tion

that,

could

of chymotrypsin

not

with

Adsorption

be anticipated

faces

can be a problem, storage

released

and Swaisgood all

that

(unpublished

The regeneration following

reductive

significant structure reduced

breakthrough chymotrypsinogen potential

from

which have

during

acid

functional

that

A in solution

action

to form SG-CTGn to glass

solutions.

surHowever,

zymogen was effectively

analysis

of a 30-day-old with

Gdn*HCl

Gdn.HCl.

Cho

effectively

beads. three-dimensional

structure

chymotrypsinogen

represents

in our understanding of the The air-reoxidation

proteins. enzymatic

reaction

had been washed

of matrix-bound

of the matrixof CTGn prior

and binding

demonstrated

activa-

of rr-chymotrypsin.

H20, adsorbed

succinyl-glass

of biologically

in multi-chain

1.4 % of the

in cold

most

innxobilization

of a did

zymogen,

the autocatalytic

protein,

of the amino

denaturation

that

in the case of dilute

data)

protein

indicate

beads

immobilization The possibility bound

a good source

CTGn is a basic

particularly

of an aliquot

adsorbed

covalently

in solution,

to the glass

of the SG-CTGn beads with

reported form.

By minimizing

may provide

as shown by comparison

preparation removes

since

first

The results

generated trypsin

the of the

to trypsin.

of zymogen

could upon

occur.

molecules

activation

is

to the enzyme

arrangement

CTGn was accessible

to its

this

activation

due to spatial

by trypsin

bound

knowledge,

subsequent

achievement of tertiary of denatured, fully

had resulted in a maximum recovery of In the present study, 53 % of activity (2).

1072

a

Vol. 48, No. 5, 1972

the

zymogen's

potential

immobilization function reduced

BIOCHEMICAL

esterolytic

of the

appears

activity

polypeptide

of intermolecular

which

bonds.

suggesting

ment of functional tions

of residues

an even wider eration the

apparent

proteins

the the

of proteins

primary

sequences

previously

in artificially

applicability

of the recent A (11)

zymogens

exemplified

of enzymatic among the

of growing bonds

shown.

formation in the polypep-

earlier

and the achieveinterac-

may now be extended Moreover,

immobilized somewhat

Thus,

thermodynamic

(10)

successful

to the

and the

complexes. upon the

of

in recovery

may be achieved

of disulfide depend

such as ribonuclease

of proteolytic

effect

conditions

of interactions

to aggregation

polysomal

location

than

conformations

under

of the attachment

structures

comprising

array

of active

that

tertiary

lead

A similar

of proteins by virtue --in vivo synthesis tide chains to ribosomes in the active observations

The increase

due to the avoidance

molecules

disulfide

was recovered

chains.

to be primarily

chymotrypsinogen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

solid-phase

to

such regen-

proteins

extends

synthesis

more complicated

of

class

by chymotrypsinogen. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Epstein, C. J., and Anfinsen, C. B ., 2. Biol. Chem. 237, 2175 (1962). Brown, J. C., and Horton, H. R., --Proc. Sot. &. Med. 141, in press --Biol. (1972). Biophys. Acta 258, 675 (1972). Cho, I. C., and Swaisgood, H. E., Biochim. Robinson, P. J., Dunnill, P., and Lilly, M. D., Biochim. Biophys. Acta 242, 659 (1971). Blow, D. M., Birktoft, J. J., and Hartley, B. S., Nature 221, 337 (1969). "Properties of Selected Commercial Glasses," Form B-83, Corning Glass Works, Corning, New York. Copyright 1949. Ellman, G. L., a. Biochem. Biophys. 82, 70 (1959). Hummel, B. C. W., Canad. 2. Biochem. Physiol. 37, 1393 (1959). Lilly, M. D., Horn=. E., and Crook, E. M., Biochem. 1. 100, 718 (1966). Epstein, C. J., Goldberger, R. F., and Anfinsen, C. B., Cold Spring Harbor m. Quant. Biol. _28, 439 (1963). Gutte, B., and- Merrifield, R. B., 2. --Amer. Chem. Sot. 9l, 501 (1969).

1073