167

Clinica Chimica Acta, 0 Elsevier/North-Holland

92 (1979)

167-175

Biomedical

Press

CCA 9958

EVALUATION OF A RAPID, SENSITIVE AND SPECIFIC ASSAY FOR THE DETERMINATION OF COLLAGENOLYTIC ACTIVITY IN BIOLOGICAL SAMPLES

M.F. LEFEVERE

*, G.A. SLEGERS

and A.E. CLAEYS

Laboratorium uoor Analytische Chemie, Faculteit Farmaceutische Wetenschappen, Rijksuniversiteit Gent, Akademisch Ziekenhuis, De Pintelaan, 135, B-9000 Gent (Belgium)

(Received

August

21st, 1978)

Summary Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histoly ticum collagenase have been investigated.

Introduction Assays for collagenolytic enzymes include a variety of methods. Some are based on the change of physicochemical properties of collagen gels (viscosity and gel-lysis measurements) [ 11, other methods involve synthetic peptide substrates [ 21, or solid collagen fibrils whose breakdown is monitored by the assay for free amino acids or for hydroxyproline [ 31. A separate group is formed by the radiochemical methods in which the release of 14C or 3H activity from a labeled collagen substrate (soluble or gel form) is followed. In a study on alkaliburned rabbit corneas, we needed a very sensitive assay, sufficiently specific to eliminate noncollagenolytic protease activity, and easily adaptable to large amounts of samples. Several methods were compared, and finally a radiochemical assay using [3H]acetic anhydride labeled collagen as substrate was selected as the most practical, and was investigated further. From the physicochemical methods, only the capillary collagen gel lysis-test, * To whom correspondence

should be addressed.

168

developed by Berman et al. [4] could be considered from the point of view of sensitivity. This assay gives a semiquantitative measure of enzyme activity. With the commercially available soluble collagen prep~ations, however, we could not reproduce the results obtained by Berman and coworkers, while the preparation of soluble collagen from the tendons of calf or rat tail, or from the skin of guinea pigs, requires a rather cumbersome and time-consuming extraction procedure, which we found less practical for our purpose. Another disadvantage is the very long incubation period (600-1800 h) needed to obtain the sensitivity required. Synthetic substrates can be used in the analysis of pure Clostridium histoIy ticum oollagenase bul are unsatisfactory for collagenase of questionable purity or different specificity [ 21. This was the case in our experiments. The procedures using native insoluble collagen fibrils and a calorimetric detection of breakdown suffer from lack of sensitivity 131. Instead, fluoresamine (Fluram @), Roche) could be used for a more sensitive fluorimetric detection of the amino acids formed after the digestion of a collagen gel or collagen in solution. The lack of specificity remains, however, as other protease enzymes can liberate amino acids from non-collagenase-specific regions of the collagen molecule. Radiochemical methods. Several procedures are described in the literature to prepare a labeled collagen substrate. These methods can be divided into two main categories: in vivo and in vitro labeling. a. In uiuo labeling. Mostly this is done by injecting guinea pigs with [‘“Clglycine or a mixture of [3H]proline/[3H]hydroxyproline [ 5--71 followed by extraction of the skin. The acid or neutral soluble collagen thus obtained has well defined properties, but a rather low specific activity. Moreover, the isolation procedure is very laborious, as cited above, and, due to the number of guinea pigs and the large amounts of labeled material needed, this type of substrate is rather expensive. 6. In vitro labeling. In this category, again 2 possibilities exist: (1) culture of a collagen producing tissue in a nutritional broth containing 14C- or “H-labeled glycine or proline [S] ; (2) chemical labeling procedures. First we tried the culture method. The calvaria from twenty 17-day-old chick embryos were removed aseptically and incubated for 24 h in 20 ml Eagles medium containing 50 r_Ci (130 pCi/mg) uniformly labeled [‘“Clglycine. The isolation procedure was exactly as described by Robertson et al. [ 81. We found it difficult to obtain both the labeled and carrier collagen in soluble form after the various precipitation, centrifuging and dialysis steps. Most of the activity was retained on the celite filter. Chemical labeling of previously isolated and purified collagen has many advantages. The purity and properties of the substrate can be determined prior to labeling, the methods are less laborious and less costly. Labrosse et al. [3] use 3H1S to label lyophilized acid soluble rat skin collagen. The procedure developed by Gisslow and McBride [2] enables the preparation of large amountx of substrate of a high specific activity in a very simple way. Acidsoluble calf skin collagen is acetylated by a solution of [ l-‘4C]acetic anhydride in benzene at pH 8.0. The label was proved to be in the collagenase-susceptible part of the collagen molecule, which was not denatured by the acetylation pro-

169 TABLE

I

SPECIFIC Labelmg

ACTIVITIES

FOR VARIOUS

Collagen

procedure

In viva [I 4 ClGlycine [14ClGlycine [3HlProline/ [3H]hydroxyproline In wtro [14C]Glycme m nutritional broth culture of chick calvaria 3 Hz S method [l-14ClAcetlc anhydrlde * Final concentration

LABELING

of the labeled

PROCEDURES Specific

fraction

Ref.

activity

Gumea pig skin, neutral salt soluble Guinea pig skm, acld soluble Rat skin, neutral salt soluble

2-3 X104 cpmlmg 2.5 X IO3 cpm/mg 3 X lo4 dpmlmg

9 1. 5 7

Chick calvarux + rat skin collagen (carrier) Acid-soluble rat skin Acid-soluble calf skin

2 x 105 cpm/mg

8

solutmn

(+ 20 ml) in which

*

6.3 X lo5 dpm/mg 5-6.5 X lo5 dpm/mg

3 2

50 mg carrier was dissolved.

cedure. We used in our experiments a variant of this method and obtained an even lo-fold higher specific activity using [ 3H] acetic anhydride. Table I gives an idea of the specific activities obtained with different labeling methods. Materials and methods Chemicals Acid soluble calf skin collagen was purchased from Sigma, as well as the Clostridium histolyticum collagenase (Type I Fraction A, 150 U/mg), Trypsin solutions were made from Crystalline Trypsin NOVO (45 FIP-units per mg). [ 3H]Acetic anhydride (100 mCi/mmol) was supplied by the Radiochemical Centre Ltd., Amersham. All enzyme solutions were prepared in 0.05 M Tris-HCl buffer (pH 7.5), containing 0.005 M CaCl,. The same buffer was used in the assays. The scintillator fluid contained 50 mg POPOP (1,4-di- [ 2( 5-Phenyloxazolyl)] -benzene), 5 g PPO (2,5-diphenyloxazole) and 100 g naphthalene in 1 1 dioxane. Acetylation of collagen 205 mg acid soluble calf skin collagen were solubilized in 90 ml 0.01% acetic acid by stirring overnight at 4°C. The acetylating agent [3H]acetic anhydride 0.98 mCi/mg, 25.5 mg in 3 ml benzene, was added dropwise over a period of 3 h under continuous stirring. Immediately before starting the addition of acetylating agent, the pH of the collagen solution was adjusted to 8.0 with 1 M K,HPO, and 1 M NaOH, and maintained at 8.0 by automatic titration with 1 M NaOH. This pH control is critical for the reaction [ 21. One hour after all the acetic anhydride had been added, the reaction was stopped by adjusting the pH to 4.0 with glacial acetic acid, and the benzene was removed by a gently bubbling nitrogen stream through the reaction medium for 4 h. The acetylated collagen solution was dialyzed against a volume of 1 1 of distilled water, which was continuously refreshed at a rate of 0.5 l/h. 60 1 of distilled water were used in total to remove the dialyzable activity. Finally, acetylated collagen was

170

dialyzed against 4 1 of 0.01% acetic acid for 24 h. During the complete procedure (acetylation and dialysis) the temperature was kept constant at 5°C. The acetylated collagen solution was stored at 4°C. Absolute activity analysis As the counting efficiency showed a marked difference even between vials from the same assay, a correction for quenching was necessary. In the Packard 3380 Tri-Carb liquid scintillation spectrometer, an automatic external standaridisation system (AES) is built-in. After an initial sample count an 241Am226Ra source is automatically moved from a shielded location into close proximity with the sample and counted together with the sample. Results of the second standardisation count are subtracted from those of the first to give net counts induced by the source in each window. The external standard ratio is calculated by dividing the net counts in one window by those in the other. The counting efficiency of unknown samples is established by comparison of the external standard ratio to a calibration curve prepared from a series of quenched samples. Therefore, to a series of vials representing different quenching levels, 50 ~1 of 3Hz0 with a specific activity of 101 570 dpm were added. Each vial was counted for 5 min. From the net cpm the % counting efficiency could be calculated. This gave us 22 AES ratios in the range 0.45-0.73 and the corresponding counting efficiences (Fig. 1). dpm (decay per minute) could be calculated from the formula dpm _ net counts per minute (cpm) % counting efficiency

x 100

Assay of the acetylated collagen 25 p 1 of the acetylated collagen solution were counted after dilution to 10 ml with scintillator solution. From the net cpm and external standard ratio, the dpm and specific activity of the labeled product were calculated. To test the specificity of the substrate, the response for a solution containing 50 pg trypsin in 25 ~1 Tris-HCl buffer, was compared to that of standards, ranging from 0.094 to 1.5 pg Clostridium collagenase and a blank (Tris-HCl buffer). Set up of the collagenase assay Test solutions were made containing 1.5, 0.75, 0.375, 0.187 and 0.094 pg Clostridium collagenase in 25 ~1 Tris-HCl buffer. All assays were performed at 37°C but the amount of substrate and incubation times varied. In type I assays, 25+1 aliquots of the enzyme solutions were transfered to a test tube containing 250 ~1 (+570 pg) labeled substrate and 500 ~1 Tris-HCl buffer. The samples were incubated for 150 and 250 min and then the remaining substrate was precipitated by adding 100 ~1 4% phosphotungstic acid and 100 ~1 4 M HCl. After 15 min digestion, the samples were centrifuged at 8400 X g for 5 min. From each tube two aliquots each of 100 ~1 supernate were counted. In type II assays, the same 25 ~1 of test-solution were added to 100 ~1 (?230

171

pg) substrate solution and 250 ~1 buffer. After incubation of three series for 30, 200 and 270 min, respectively, the samples were defecated by adding 50 ~1 of phosphotungstic acid solution and 50 ~14 M HCl, and handled the same way as for type I. From each supernate, again two lOO+l aliquots were taken and counted. Study of the influence of salt concentration, tion on collagenoly tic activity

low temperatures

and lyophilisa-

We wanted to know if there was any influence of low temperatures and lyophilisation on enzyme solutions with various salt concentrations, as they would occur in our cornea extracts. Thus we prepared enzyme solutions with 0.05 M; 1.55 M and 3.55 M salt concentration containing, respectively, 0.015 pg/pl; 0.015 pg/pl and 0.019 pg/r.ll Clostridium collagenase, 0.05 M Tris-HCl buffer, and, in solution 2 and 3, NaCl to adjust the molarity. Two blanks were prepared also, one containing 0.05 M Tris-HCl and the other 4 M NaCl. Each solution was divided into three parts, one stored at 4°C the second was kept at -20°C for 3 days, and from the third, 250 ~1 were frozen in liquid nitrogen and lyophilized. After being kept for 3 days under the lyophilized form, the original volume of 250 ~1 was restored with distilled water. Finally from each solution, two samples of 25 ~1 were taken and analyzed as described for assay type II. The incubation time was 120 min at 37°C. Results and discussion 1. cpm to dpm conversion

For the 26 corresponding external standard ratios and percentage efficiency values of the Absolute Activity Calibration, the linear and exponential relationships were calculated. For the linear regression equation (y = 60.27x - 4.83), we found a correlation coefficient r = 0.91. Although the exponential relationship y = 9.25 ez.Oox) had a somewhat better correlation (r = 0.92), no improvement in the assay correlations was found when percentage efficiency, read from the exponential instead of the linear AES calibration graph, was used to calculate dpm. For the given range AES 0.45 to 0.73, both the linear and exponential calibration graphs may be used (Fig. 1). Direct regression analysis on assay values expressed as cpm, gave a markedly lower correlation for the assay calibration curves, due to the above-cited difference in counting efficiencies between vials from the same assay. 2. Properties

of the acetylated

collagen substrate

We found a specific activity of 337 370 dpm per 25 ~1 labeled solution (corresponding to 57 pg collagen). This gives us a specific activity of 5.93 X lo6 dpm/mg collagen, or about lo-fold higher than the specific activity that could be obtained using [ 14C]acetic anhydride. The specificity of the acetylated substrate towards collagenolytic activity was shown by the response to an excess of non-specific enzyme, trypsin. 150 min incubation of 50 pg trypsin released an activity of 11400 dpm. This corresponds to the confidence interval limit for 0.15 pg Clostridium histolyticum collagenase (Fig. 2).

‘1. counting

ltt icicncy

35

3a

25

20

+ IDe4

0.5

0.6

0, 7

Fig. 1. ob counting efficiency vs. AES-ratio. Linear relationship exponential relationship (- - - - - -), s = 9.25 e2.00~: r = 0.92.

Flocculation 2-3 months. be lyophilised.

AES -ratto (-

), y = 60.27.~ --- 4.83; r = 0.91,

occurs if the acetylated collagen is stored in solution at 4’C for If the labeled substrate is to be stored for longer periods, it must

3. Evaluation of assay parameters All assays were performed at 37’C on soluble collagen ing concentration (0.74 pg/pl and 0.61 pg/r_ll for assay

substrate in a saturatI and II, respectively)

[21. We wanted to test the influence of total amount of substrate and incubation time on the detection limit. The lowest amount of enzyme still giving a response significantly higher than the blank was calculated from a t-test at level

65.

activity as dpm xt03

55

45.

35,

475

,

1.5 mzym* QmovIs Irg protrin

Fig. 2. Calibration curve for assay type I, 570 pig substrate and 150 min incubation fidence band around the regression equation,

time. (- - - - - -) eon-

in which est s.e. (5,) = estimated standard error on predicted signa yg corresponding to a given sample concentration x0, and o = standard error of estimate for the regression equation. 11 420 dpm = activity found for 50 fig trypsin.

TABLE11 CALIBRATION AND LOWEST CALCULATED

CURVES,

CORRELATION

DETECTABLE FOR VARIOUS

Amount substrate

Incubation time

wg)

fmir0

AMOUNTS AMOUNTS

COEFFICIENTS,

STANDARD

OF CL. HISTOLYTICUM OF SUBSTRATE AND

Calibration

curve

ERRORS

OF ESTIMATE

COLLAGENASE (150 INCUBATION TIMES s.e. *

U/mg) AS

Lowest detectable amount wg)

570

150

570

260

230

30

230

200

230

270

Y ~46 354(xi-a+18 282 r = 0.9990 y = 59147(.r-?i)+21693 r = 0.9971 y =20332(xj-Zj+10482 r = 0.9761 Y = 51143(Xi--x7+23007 r = 0.9908 ~~65 848(xi_ZJ+ 41931 r= 0.9792

* s.e., standard error of estimate on calibration ** At level of significance 0.01.

curve.

**

604

0.05

1350

0.09

1270

0.23

3700

0.27

3784

0.04

174

0 Fig.

3.

strate. 230 (X -X):

Calibration 150

min

,Ug substrate. 230

graphs

as calculated

incubation

time

30

1.5

0.75

0.375

min

/.~g substrate.

(‘1

for

incubatmn 270

various

-~1);

min

time

570

substrate ~g

4):

(C

incubation

time

amounts

substrate, 230

260

mrym.

and

amoun(, plJ prottln

incubation

min

I.rg substrate,

incubation 200

mm

times.

570

time

(a--

incubation

~.rg sub*): time

(m-n).

of significance of 0.01. Table II lists the linear relationships found, with corresponding correlation coefficients, standard errors of estimate and detection limits. The assays were linear up to concentrations of 0.75 pg enzyme. For higher amounts of enzyme a saturation can be observed (Figs. 2 and 3). The use of higher amounts of substrate (570 1.18, assay type I) allows a sensitive detec-

175

TABLE

III

ENZYME ICUM

ACTIVITY

(AS

COLLAGENASE

LYOPHILIZED

% OF

INITIALLY

SOLUTIONS

FORM

FOR

RELEASED

OF

dpm)

VARIOUS

SALT

AFTER

STORAGE

CONCENTRATIONS

OF

CL.

AT

-2O’C

HISTOLYTAND

IN

3 DAYS Activity

Enzyme

salt

Initial

Activity

after

concentration

concentration

activity

storage

at -20°c

wg/w

(M)

(%)

for

after

lyophilisation

3 days

(%)

(%) 0.015

0.05

100

91.7

59.5

0.015

1.50

100

96.1

49.5

0.019

3.55

100

93.8

40.9

tion, which is not improved by incubation times longer than 150 min. To obtain the same sensitivity, much longer incubation times are needed if lower amounts of substrate (230 pg/assay II) are to be used. There was little or no influence of the salt concentration on enzyme activities and blank value. Storage at -20°C showed a slight decrease of enzyme activity (from 4 to 8%), but a drop of 40 to 60% was observed after lyophilisation. This effect was even more pronounced at higher salt concentrations (Table III). If samples have to be concentrated after a purification step (gel chromatography) one should keep in mind that lyophilisation will decrease the enzyme activity and that the salt concentration of the eluent should be kept as low as possible. Acknowledgement We wish to thank Professor H. Steyaert (Seminarie voor Waarschijnlijkheidsrekening en Mathematische Statistiek, State University of Ghent), for his advise on the statistical interpretation of the assay results. References Seifter.

S.

Vol.

pp.

19,

G&slow. Berman.

Harper,

613435,

M.T.

Labrosse, Nagai.

and

and

K.R.. M.B.,

Y..

Kaufman,

E.

McBride,

Lientr,

I.E.

Manabe,

Lapierre. E.J.,

(1970)

Methods

Academic B.C.

C.M.

and

Gross,

M.J..

Biochem.

P.A. P.F.

J. (1966)

Mechanic.

Enzymology

(Colowick,

S.P.

and

Kaplan,

N.O..

(1976)

(1973)

68.70-18 Anal.

Anal.

Biochemistry, G.L.

and

Biochem.

Biochem.

70, 54.

218-223

522-534

3123

Goldhaber.

P. (1965)

Proc.

SW.

ExP.

Biol.

120,632&33 Sakamoto,

S.P.,

Robertson,

P.B.,

Vaes.

G. (1972)

Goldhaber, Taylor. Biochem.

P. and R.E.

and

J. 126.

Glimcher, Fubner, 275-289

eds.),

York

Anal.

Hargrave, Davison,

in

New

(1975)

and

R. and

Glimcher,

Press,

M.J. H.M.

(1972)

(1972)

Proc. Clin.

Sot.

Chim.

Exp. Acta

Biol. 42.

Med.

4345

139.1057-1059

Med.