Simple quantitative test for measuring collagenase activity

ARCHIVES

OF BIOCHEMISTRY

Simple

AiXD

Quantitative

BIOPHYSICS

92,

Test for

l&-1%

Measuring

HOWARD From

the Product

Collagenase

Activity

TINT

Development Division, Received

(1961)

Wyeth July

Laboratories,

Radnor,

Pennsylvania

14, 1960

A linear relationship was observed when the logarithm of collagenase concentration was plotted against per cent weight reduction of refined, undegraded collagen as substrate. The slopes for 19 regression lines were determined, based on three to six observations each. From the mean slope, a nomograph was constructed for determining the amount of collagenase required to digest 50y0 of a standard substrate in 30 min. at 40°C. (EDso), providing an activity unit in a simplified and rapid assay of this enzyme.

collagenase shares w&h a large group of enzymes the capacity for hydrolyzing denat,ured collagen (l-4). It is unique in it,s ability to &tack native collagen (5). The only confirmed sourcesof collagenuse,filtrates of Clostridium welchii and Clostridillm histolyticum, contain various other proteolytic enzymes including prot#einasesand peptidases (1-8). Thus, t.he assay of collagenase activit8y requires a highly purified, native collagen sub&rate. Distinct collagenase activity x;as first demonst,rat8edthrough its effect on rabbit muscle. When exposed to filtrates of Clostridium welchii, the muscle swelled, became opaque, and showed other changes similar t,o those occurring in gas gangrene (9, 10). The residual muscle was devoid of reticular framework, but, was otherwise unaltered (11, 12). Early assay procedures were based on this observation; t#hedegree of necrosis or smelling, in intact, or isolat,ed muscle, was estimated visually. An alt,ernative procedure involved measuring t’he amount of ant’iserum required to inhibit collagenase activity (4, 13). Various yuant#itat,ive procedures for determining the amount of collagen digested have been int,roduced. These include measurements of t,he soluble nit’rogen released (2, 6, l-2), free amino groups liberat,ed (a), 154

and hydroxyproline solubilized (15). An alt,ernat,ive reliable procedure is the measurement of decreased viscositBy during t,he digestion of ichthyocol, an acid-soluble collagen (16,l’i). These more refined methods have largely replaced earlier ones based on changes in the consistency of t)he subst#rate (13). Measurement of weight, has the advantage of simplicity and facilitates assaying large numbers of samples. The present report’ describest’he development8of a simple quantitat,ive test, based on a nomograph relat8ingsubstrate weight lossand collagenase concent,ration. MATERIALS

AND

NIETHOUS

SUBSTRATE

Refined, undegraded collagen was prepared from bovine t,racheal cartilage. After manual cutting and shredding, the material was extracted, in the cold, wit.h dibasic sodium phosphate followed by pot,assium chloride, to remove soluble proteins and mucopolysaccharides. It was washed thoroughly with water, dehydrated with alcohol, and vacuum dried at room temperature. To assure the cont,inuing integrity of the substrate, aliquots were removed at various intervals during the procedure and subjected to standard incukat,ion with trypsin; weight losses were negligible. ENZYME

Collagenase \vas prepared from tridium histolyticum. The medium

filtrates of (Floswas casein hy-

COLL.kGEKASE

drol~~zutr containing defatted beef heart, phosm phates, nxtgnesium sulfate, rysteine, tryptophnn, suwinate, cnlciIm~ pantothennte, pyridoxine, rihoAfter sterilization, thioglyflnvine , :rnd glucose. colatc w:ts added; the pH of t,he medium XLS i.i7.5. The inoculum was a seed culture of strain 17(&i maint:tined in the :~bovc medium wit bout gl~~cow. FlItids were harvested after 16-18 hr. iw culxrtion :tt 3i”C., ant1 filt,ered t.hrongh asbestos kr~suction. The reli:lhility of the nss:~v procetllwe was tested wit 11 crlide filt,rat,es, filtrates dialyzed against distilled water or appropriate buffers, anti conccnt rates preparcd by Iyophilization or meth:1r101 prwipit;ition.

ACTIVITY

155

‘OOy ao5- 605 2 E 40: " i;i 320 69

50.nip. portions Iksicc~atcd, of unifornil~ shrrdd~d collagen sul)strat,e were placed in 50-1111. Erlrnmeyer flasks rontaining 10 ml. collagenasc diluled in hufferrtl sulinc. In preparing vctrious dilrltions, a11 ionic strength of 0.2 WE mnintaincd; the diluent \V:IS bufleretl at pH 7.GT.2 with phosplute or borate htlffer. The flzks WPI'C incuk)ated, with nirch:mic:L1 swirling, :rt, 40°C. for 30 min. Residual coll:~ge~~ \V:LY sedimented by centrifugntion, axshed twiw with distilled water and twirr q-it h acetow, tlrird for 30 min. ut 105”C., :md weighed. h control sample withol~t enzyme was carried through t IIP same procedure. The weight loss of the digested sample was determined b? conrp:lrisoii kvit,h the cont,rol.

A liuwr wlationship was ohtaiued 1)~ plott,ing pw wuf~ weight, reduction against logarithm of milliliters Of colla~cilasr prcparatioll. The slopes of the regression Cues for various snnlpl~s appeared to he coiistatit~ (Fig. 1). X total of 10 titmtious was performed ; the numhcr of poiuts ohtaiiitd varied from :3 to 6. The slope of the regression liue was determined for each wt of data. (‘l’al,le I). The Ill~‘atl slope \\-as 45.1 with :i stand:wd dmiatiou of 5.7. Usillg the mea11 slope, a standard equation w;w deri\-cd for dvternliuiug the volume of c~ollagenasc~ requitwl to digest XI 7; of the s1:~11dwd suhstratr (KZ&i:

whr~rc .I‘ is thr \-olumr: of cwllageuasc used, aucl yy is the per wut \veight wductiou olwx\-cd. In Tahlc II tht results of a typicsal titration are rwordml, and the El& is detcriiiiiird from the stnudard formula. It will

II -O!S -0.6

-0.41 -0.2/ LOG.

0, ML.

FIG. 1. ASS:I~S of folu collagennse.

012

0!4

COLLAGENASE

different

1)rcpnrations

i i 1 ( ~ ~

!

3 4

18

'

3

10

Standard ation

I

6 3 3

I

54.6 39.2 49.4 ~

3

AIe:tn dcvi-

3i.i 30.4 40.5 (

41 .?I

45 1 B.7

of

Per cent variation hetvv?en A and R

Slope

9 10 11 12 13 14 t5 16 17

( 1

0.6 I

I I1

2.52 2.70 2.26 2.48 4.1!) 3.81 1.50 1.55 1.49 t.45 1.41 1.45 4.05 3 .90 O.Si 0.86 1.26 1.22 0 o-4 ) 0.05 2.71 2.82 0.02 0.03 0.03 0.0:3 2.33 2.51 0.58 0.57 0.38 0.50 3.77 3.16 1.10 5.89

~ 35.42 ,69

1 :

1 (

1 1 1

+i.l +9.i -!I. 1 +:?I 3 -2.i +2.s -3.i -1.1 -3.2 +25.0 +-I.1 +33 .3 0.0 ti.2 -1.; f31.6 -19.3 -11 .!I -8.0

156

TINT

The resuhs are compared in Table I. The widest, variations were observed in t’itrations involving only t,hree points. In these cases, t,he slope of t.he regression line is greatly afPer cent Per cent fected by single aberrant, observations, when log EDao variation Collagenase ~ weight ~ reduction from mean t,he met,hod of least squares is applied. The / __.I~ e&mates obtained from t,he nomograph Ml. ’ log. ml. j compared well with those derived by appli0.5 -0.301 ' 15.4 0.460 i-8.2 cation of the Aandard formula. 29.9 0.442 j-4.0 1 .o 0.000 0.429 +0.9 Titrat,ions 1 and 2 (Table I) are repeats of 2.0 : 0.301 ~ 42.2 ~ 0.369 -13.2 4.0 0.602 60.6 the same assay on different days; similarly, ____.__ ~~ 3 and 7 are repeated assays of another Mean log EDso 0.425 preparat,ion. It should be noted t>hat in EDso 2.66 both cases the greatest variability was observed when the least-squares method be noted that the range of variation for was used. Thus, the st#andardcurve appears single points, in this titration, is from +8 t,o give a more reliable measureof collagenase to -13 % of t,he mean value. In practice, activity. t,he average value for log EDso obtained from DISCUSSION 3 to 6 observations is used. It, has been established that, collagen On the basis of the st,andard curve, a nomograph was construct,ed for det8ermining digestion follows first-order kinet,ics over a the EDso from a single point, (Fig. 3). The wide range of enzyme and substrate conEDSO’sfor 19 tit,rat)ions mere determined by centrations (17, 18). Thus, by plotSting the two methods: (a) the method of least, logarithm of the milligrams collagen resquares; and (b) the antilogarithm of the maining in suspension against, t.ime, Gallop average value obt,ained from the nomograph. et al. (17) obtained a series of lines from TABLE

DETERMINATION STANDARD

II

OF EDjo FORMULA

FROM

‘0°711T-171TillX

- I:6 1.6

- I.2

YI

-0.8

-0.4 LOG

0 ML.

0.4

0.8

1.2

Yll

1.6

COLLAGENAX

FIG. 2. Nomographfor determining EDao value. Directions: Locate the point representingthe logarithm of the volume of collagenase used and the per cent weight reduction observed. Follow the nearest diagonal line to its point, of intersection with the 50yo line; read log EDso on the abscissa below this point of intersection. Estimated positions between the guide lines may be used.

COLLAGENASE

which t.he act’ivity of unknown preparat’ions could be determined. A similar series of curves was obtained when the logurit’hm of t,he specific viscosit,y of a suspension of ichthyocol was plotted against time. Mandl et ad. (18) observed good proport’ionality hebween t,he first-order rate constant, and the c*oncentjrat’ion of enzyme. With tirnc and substrate csonrentrat.ion constant, Gallop et al. (I 7) and Grant’ and Alburn (19) obt,ained a curvilinear rrlationship by plott,ing milligrams of collagen dissolved against micrograms of collagellasc. The relationship was approximat’ely linear unt,il -l mg. collagen had been dissolved. In earlier work, Bergman11 (20) not,ed that the early part of collagen digestion followed his formula for dig&ion of gelat)in: IJ = Kt . y’;, \vhere R: = per (sent enzyme ~~~ncentrat,ion; K is a con&ant; t = time; and y = per caent digest’ion. Our results indicsatr that’ the relationship between the logarithm of the amount of collagenase and per cent digestion is linear over the entire digestion of 60 mg. cwllagen. The apparent diwrepanciw arc explained by comparison of the arithmetic plot’s of the various curves (Fig. :i). It will be not,ed that t,he linearity described by Gallop and Grant is satisfied by our curve up to about 5 ‘2 Bergmann’s (wrve, with tirne constant, follows ours c*losely up to about’ I.5 q?’ digestion. The establishmellt~ of a formula relating cY~llagf?Ilasc concentration and digestion, which is applicable over the entire range of the reaction, permits the construction of a nomograph for determining the collagenase collt ent of various samples. It’ should be ttmphasized that) the nomograph presented iit this report, is applicable only for our twt cwllditions. When the experimental cwndit,ions are varied, a sirnilar nomograph may be constrwt’ed, ba,sed on the obsrr\wl slope. In thta comparison of c*ollagcnase wt,ivitJy of various preparatioils, we routinely assay them in the same twt,. Thus, the reprodwihility of the results for repeat,ed tjitrat,ions has received only minor attellt,iotl in this study. The results do inditat,e, howwcr, t’hat rcprodwihlc results might tw expected if a standard substrate and tlilurllt are used. The standard formula

157

ACTIVITY

90.

4

y-45

LO‘ Y + B

FIG. 3. Comparison of arithmetic plots of ecp~ations described for relating collagenitse concent ration and per cent digestion. .l: Tint ient.ire range of reaction) ; B: Bergmann (early part. of reaction) ; (‘: (bllop cl al. and Grant, :mtl Alhurn (to digestion of 1 mg. rollngen). and the Ilomograph appear to give a more reprodwible measure of collagenase act,ivity than the least-squares method. This undoubtedly reflects errors in the slope:: of the regression lines when the least-squares method is applied to relat’irely fen- poillts. The use of the nomograph permits rapid c*alrulation of the EDso value. values determined from siuglr ~‘nso observations are, of course, considerably less reliable than those de&mined on the basis of several observations. RJ15C1values were determined from eight’ single observations for one preparation (titrations 1 and 2). The mean \raluc oht,ained from t,hesc determinations was 2.58 with a standard dcviation of 0.41. Similarly, E& values \vpre determined from ten single observations fOJ another preparation (titrations 3 and i) ; the mean \-aluc was 4.91 with a standard deyialion of 0.56. Thus, two st’andurd deviations were 24.0 ‘/;a of the mean for one preparatiort, and 28.0 (:;’ for the ot#her.

1.

hC

LENNAN,

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JIIINDL,

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