BIOCHEMICAL

Vol. 74, No. 3, 1977

THE STOICHIOMETRY

AND BIOPHYSICAL

OF INHIBITION

RESEARCH COMMUNICATIONS

OF HUMAN PANCREATIC

ELASTASE 2 BY HUMAN ALPHA,-ANTITRYPSIN Michael

Tyndall,

Corey

Department of Medicine, Martinez, California 94553 School of Medicine, Davis,

Received

November

James W. Brodrick,

Largman,

Martinez Veterans and the Department California 95616

+

and Michael

Administration of Medicine,

C. Geokas

Hospital, UC-Davis

29,1976

Summary: A 1:l stoichiometry of inhibition of human pancreatic elastase 2 by human al-antitrypsin has been determined. The molar binding ratio was calculated using the results of a titration curve for elastase 2 inhibition an experimentally determined concentration of active sites by al-antitrypsin, in human elastase 2, and an extinction coefficient calculated from ultracentrifugation studies using interference optics. INTRODUCTION Elastase ability been bling

is

unique

to hydrolyze suggested

elastin

as a cause

pulmonary

pancreatic

previously

reported

form

of this

in easily

detectable

Elucidation

of the

present

in

mechanisms

'This research Administration

endopeptidases

For this

reason,

lesions

in the

to either

isolation,

elastases

enzyme

pancreatic lung

(4).

(elastase

interaction serum

hamsters

2) is

present

as measured

elastase-mediated

the

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

(3).

We have

that

of normal

individuals (5).

protease

inhibitors

to formulate

possible

hide

tosyl-L-arginine powder; and p-NPGB,

Medical Research Service of the the California Lung Association. 857

the

injury.

3H-DIFP, 3H-diisopropylfluorophosphate*TAME, RBB-Hide, Remazol Brilliant Blue dyed p'-guanidinobenzoate HCl. was supported by the and by a grant from

resem-

by administration

by radioimmunoassay

in order tissue

Lesions

demonstrated

in sera

enzyme with

necessary

has

and characterization

We have also

of this is

or dogs

in its elastase

(2).

induced

purification,

concentrations,

of pancreatic

serine

have been experimentally

on the

human blood

Abbreviations: methyl ester; p-nitrophenyl

(1).

elastase

of two human pancreatic

pancreatic

of tissue

emphysema

of porcine

major

among the

Veterans

Vol.74,

No. 3, 1977

BIOCHEMICAL

Human al-antitrypsin ponsible (6).

for

trypsin

deficient

binding

of porcine

contrast

to previous

a,-antitrypsin careful elastase

(8)

have

trypsin

inhibitory

evidence

which

of the

severe

associated

for

by al-antitrypsin.

serine

in high

con-

al-anti-

with

premature

(7).

presented

reports,

is

of plasma

present

determined

which

to be res-

capacity

and is

A genetically

emphysema

RESEARCH COMMUNICATIONS

has been shown

specificity

has been described

and various

determination

a broad

human serum.

state

--et al

which

90% of the trypsin

of pulmonary

Johnson

a glycoprotein

exhibits

in normal

development

this

approximately

The inhibitor

centration

is

AND BIOPHYSICAL

These

indicated proteases

stoichiometry

2 to human al-antitrypsin,

a 2:l

a 1:l (9).

results

results molar

are complex

We therefore

of binding

the

stoichiometry

of

in marked between

undertook

a

of human pancreatic

of which

are

presented

in

report. MATERIALS

AND METHODS

3H-diisopropylfluorophosphate (3H-DIFP) was obtained from Amersham Bovine trypsin employed in the determination of inhibitory caSearle, Inc. pacity of a -antitrypsin was obtained from Worthington Biochemical Co. Fresh from the Martinez Veterans Administration Hospital. human plas ml was obtained Human a,-antitrypsin was prepared by the method of Pannell et -- al (11). This procedure included passage of fresh human plasma through a blue-dextran conjugated-Sepharose 4B column in order to remove serum albumin. Material eluting from this column was subjected to ammonium sulfate precipitation followed by DEAE-cellulose chromatography at pH 8.8 and at pH 6.5. The resulting a -antitrypsin migrated as a single band in discontinuous sodium dodecyl sultate polyacrylamide gel electrophoresis and inhibited 60.3 units of tryptic activity per mg of a -antitrypsin. Tryptic activity was determined weight of 53,000 daltons by measuring the hydrolysis of +AME. A molecular and an extinction coefficient of E 2so = 5.30 (11) were employed in the calcu1% lation of binding ratios. Human pancreatic elastase 2 was purified to homogeneity by the previously The enzyme migrated as a single band in analytical described procedure (4). polyacrylamide gel electrophoresis at pH 4.5 and 2.3 and in discontinuous A molecular weight sodium dodecyl sulfate-polyacrylamide gel electrophoresis. of 25,000 daltons (4) was used for calculations of molar binding ratios. An using extinction coefficient for elastase 2 of E2fo = 24.9 was determined interference optics as described by Babul &!d Stellwagen (12). Active Site Titration of Elastase 2. Determination of the active site concentration of the elastase 2 solution employed for molar binding ratios was performed by labeling the enzyme with 3H-DIFP as described by Robinson et al (13). The specific activity of the 3H-DIFP employed was determined by Eb?iing an aliquot of a solution of bovine trypsin in which the concentration of active

858

BIOCHEMICAL

Vol. 74, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sites was independently determined by titration with p-NPGB using the method of Chase and Shaw (14). To a 1 ml solution of elastase 2 (0.2 mg) in 0.05 M Tris.HCl (pH 8.0) were added 5 ~1 of 3H-DIFP (105 cpm) in dry isopropanol. The reaction mixture was incubated for 1.5 h at 370 and then applied to a 1.2 x 10 cm column of Sephadex G-25 equilibrated with the reaction buffer. The 3H-DIP-elastase 2 was eluted as a symmetrical peak and the ratio of cpm per mg A parallel reaction of protein was determined for each of the peak fractions. was performed with 0.3 ml bovine trypsin (0.43 mg, 1.75x10-8 moles/mg, deterUsing the specific activity mined with p-NPGB) and treated in a similar manner. of 3H-DIFP obtained from the concentration of active sites of the trypsin solution, the concentration of active sites in elastase 2 was calculated to be 1.60 x lo-5M. This value corresponded to an approximately 99% active solution of elastase 2, based upon an extinction coefficient of 2.49 (mg/ml)-1 and a molecular weight of 25,000 daltons (4). Elastase 2 Activity Measurements. RBB-hide is a good general proteolytic was prepared as described previously of 10 mg/ml in assay buffer consisting 25 mM CaC12, 1 mM Triton X-100.

Previous work (4) has shown that substrate for elastase 2. This substrate (15) and employed at a final concentration of 0.1 M Tris.HCl (pH 8.2) containing

Inhibition Assays. Incubation of elastase 2 with al-antitrypsin and the subsequent determinations of proteolytic activity were designed to maintain a 2 ml total assay volume containing 0.32 ug of elastase 2. Elastase 2 (0.32 pg in 10 ~1) in 10 mM sodium phosphate (pH 6.5) was incubated in duplicate with 0 to 1.48 pg of al-antitrypsin in a total volume of 2.0 ml of assay buffer. After a 45 min incubation at 230, the contents of each tube was carefully transferred to an assay tube containing 10 mg of RBB-hide. The RBB-hide assay mixture was incubated for 30 min at 370. The assay tubes were then placed in ice water, centrifuged at 6,000 rpm for 10 min at 40, and finally kept on ice for a minimal period until the absorbance of each assay mixture at 595 nm could be determined. The inhibitor-enzyme ratios at complete inhibition were determined by extrapolating the linear portion of the dose-response curve for inhibition as described by Laskowski and Sealock (16). In all cases, the linear part of the inhibition curve extended to 85% inhibition or more. RESULTS AND DISCUSSION A molar

extinction

was determined extinction finding than

found

studies.

This

elastase value

of EfT"= o 20.2 reported for porcine 0 reflects a greater content of tryptophan

in the

porcine

inhibition which

activity.

were

Although

approximately In order

human pancreatic

coefficient

Initial mixtures

for

by ultracentrifugation

probably is

coefficient

l:l,

to minimize

enzyme

experiments diluted these

the curve possible

could

were

performed

with

ten-fold

for

be extrapolated

was non-linear effects

0 than the

is higher elastase in the

(17).

This

human enzyme

(17).

approximately data

2 of $"=

at low

produced

859

concentrated assay

of elastolytic

to a binding inhibitor

by dilution

incubation

ratio

concentrations. of the enzyme

of

24.9

BIOCHEMICAL

Vol. 74, No. 3, 1977

AND BIOPHYSICALRESEARCH

moles

a ,-AT

: mole

dostase

COMMUNICATIONS

2

Figure 1. TITRATION OF HUMAN ELASTASE 2 WITH HUMAN al-ANTITRYPSIN. A constant amount of elastase 2 was incubated with varying amounts of a -antitrypsin (O-l.8 fold molar excess) in a constantvolume of 2 ml o + assay buffer as described in Methods. Following a 45 min incubation period, the mixtures were assayed for proteolytic activity on RBB-hide as described in Methods.

inhibitor

incubation

mixtures

for

the enzyme and inhibitor

were

experiment

using

curve

was performed

of elastase

inhibitor with

elastase

extinction 2 to calculate

periods,

the

enzyme with

resulting the

inhibitor

The results pancreatic

two moles for

Recently,

site

trypsin. as well

group

period, for

mole

was performed

in which When such an

a linear

inhibition

the entire this

titration

of 0.98

was non-linear,

experiments

by Johnson

of porcine

this

ratio

conditions.

Data from

and active

using

range

of

curve

was combined

values

determined

for

2 per mole

of

of elastase

indicating

provide

2 and al-antitrypsin.

reported

human trypsin

1.

performed

15 min incubation that

the reaction

of

was not complete.

of these

elastase data

in Figure

experiment

curve

assay

was obtained

a binding

When this

under

were

a 45 min incubation

coefficient

al-antitrypsin.

tative

as shown

experiments

incubated

2 by al-antitrypsin

employed,

the

assay,

evidence This

--et al

(8)

as bovine

has suggested

result

who found

The same group

also

chymotrypsin that

leukocytic

860

for

a 1:l

complex

contradicts that

reported

the quanti-

al-antitrypsin

bound

a two to one binding

to al-antitrypsin elastase

for

is

(18). bound

by

Vol. 74, No. 3, 1977

BIOCHEMICAL

al-antitrypsin

complex

in a 1:l

results

with

complex

for

reported have

isolated

a 1:l

is

molar

binding

were

Kress

retracted

their

and Laskowski Moroi

of amino

a 1:l

(9)

and human al-antitrypsin

on the basis

earlier

to demonstrate

by q-antitrypsin.

trypsin

ratio

also

presented

hand,

trypsin

of bovine

authors

have

and Yamasaki and have

acid

composition

(10).

essentially

to previous

100 per

obtained

result

but no data

of bovine

a complex

These

(19).

On the other

enzyme.

binding

In contrast

were

trypsin,

either

a 1:l

calculated data

porcine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which

suggests

probably

twice

cent

work,

active

demonstrate

that that

the present

the concentration suggested

employed

highly

purified,

2 and human aI-antitrypsin.

elastase a 1:l

study

binding

of enzyme by the

of al-antitrypsin

by Johnson

et -- al

inhibitor.

in human blood

Data This plasma

(8).

ACKNOWLEDGEMENT We wish to thank the ultracentrifugation

Dr.

Jeff Howlett studies.

and Ms. Boihan

Yee for

performing

REFERENCES 1. Partridge, S.M. and Davis, H.F. (1955) Biochem. J. 61, 21-30 Loeven, S.A. (1969) J. Atherscler. Res.T9-390 i: Hayes, J.A., Korthy,-A., and Sniderx.L. (1975) J. Path. 117, 1-14 4. Largman, C., Brodrick, J.W., and Geokas, M.C. (1976)BiTTchemistry 15, 2491-2500 5. Geokas, M.C., Brodrick, J.W., Johnson, J.H., and Largman, C. (1976) J. Biol. Chem., in press 6. Heimburger, N., Haupt, H., and Schwick, H.G. (1971) Proc. Int. Res. Conf. on Proteinase Inhibitors, Fritz, H. and Tschesche, Hzedsxpp r--2i, i%-lin. Walter de Gruvter 7. Laurell, C.B. and Eriisson, S. (1963) -*Stand J -*Clin 15 _-*Lab -*Invest -3 132-140 8. Johnson, D.A., Pannell, R.N., and Travis, J. (1974) Biochem. Biophys. -Res. co mmun. 57, 584-589 9. KF ess, L.F. and Laskowski, M. e Inhibitors, ed. Fritz, H., et Moroi, M. a nd Yamasaki, M. n9 1:: Pannell, R., Johnson, D., and -5445 12. Babul, J. and Stellwagon, E. ( 13. Robinson, N.C., Neurath, H., and Walsh, K.A.3)Biochemistry 12, 420-425 14. Chase, T. Jr *. and Shaw, E. (1970) Methods Enzymol. l9-, 20-30 15. Rinderknecht H., Silverman, P., Gebkas,.C., and Haverback, B.J. (1970) E. Chim. Acta 28, 239-246 16. LaskowK M. and Sealock, R.W. (1971) Enzymes, 3rd Ed., 3, 376-473 B.S. and Shotton, D.M. (1971) Enz mes, 3rd Ed., 3, 323-373 17. Hartley, 18. Travis, R+) J., Johnson, D., and Pannell, z Srposiu; V, Proteinase Inhibitors, ed. Fritz, H., --et al, pp - , ew Yor , SpringerVerlas 19. Baugh; R.J. and Travis, J. (1976) Biochemistry 12, 836-841 861