Isolation and purification of human type I procollagen by adsorption to glass beads

ANALYTlCAL BIOCHEMISTRY 96, 433-447 (1979)

Isolation and Purification of Human Type I Procollagen by Adsorption to Glass Beads

STEVE GERARD AND WILLIAM M. MITCHELL

Department of’ Pathology, Vanderbilt University School of Medicine* Nashville, Tennessee 37232

Received November 16, 1978

Procollagen from the culture medium of human foreskin fibroblasts is efficiently adsorbed on controlled-pore glass (CPG) beads. Elution of adsorbed protease( capable of procollagen degradation, is accomplished with 1 M phosphate. This allows subsequent purification steps to be accomplished without detectable degradation of the high-molecular-weight procollagen form which is subsequently eluted with 1 M Tris. Analysis of the Tris elution fraction from CPG beads by sodium dodecyl sulfate-agarose-polyacrylamide electrophoresis on 2% gels indicated that the majority of protein is types 1 and III procollagens and partially processed intermediates. Types I and III procollagens were separated by DEAE-cellulose chromatography, and presumptive undegraded type I procollagen was resolved from processed forms by molecular sieve chromatography in 1 M CaCl, on agarose beads. The high- molecular-weight type I human procollagen isolated by this method was found to contain both amino and carboxy-terminal propeptides. Two o1 and one cu2 procollagen chains, disulfide bonded via the carboxy-terminal propeptides, are present per molecule. This procedure represents an efficient and relatively rapid method for preparing human procollagen in sufficient quantity for detailed chemical analysis.

Tissue culture has been widely used as a source of procollagen, the precursor form of tropocollagen. Procollagen conveniently accumulates in the culture medium although isolation of intact procollagen is hampered by the concomitant presence of proteases which are active in the conversion of the soluble precursor to an insoluble tissue matrix. This problem has been most fre- quently dealt with by omission of serum from the culture medium (l-5) which greatly reduces procollagen protease activities or by including a variety of protease inhibitors in the various stages of purification ( 1,s - 8). The methods for procollagen isolation are laborious and time-consuming, including that described by this laboratory for human procollagen secreted by fibroblasts in culture (1).

We report herein a rapid and relatively simple method for the isolation of undegraded type I human procollagen. The

method takes advantage of the usually frustrating property of procollagen to adsorb to surfaces (1,9, lo), in this case to controlled pore glass (CPG)’ beads. Isola- tion and purification by adsorption to CPG beads and chaotropic ion elution has been previously employed for the preparation

’ Abbreviations used: CPG, controlled pore glass; Hepes, N-2-hydroxyethylpiperizine-N’-2-ethanesul- fonic acid; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; Tris, tris(hydroxy- methyI)aminomethane; DEAE, diethylaminoethylene; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; TEMED, N, N, N’, N’ ,-tetramethylethylene- diamine; DTT, dithiothreitol; TLCK, tosylyslchloro- methyl ketone; BSA, bovine serum albumin; proa, single collagen chain precursor with both amino- and carboxy-propeptides; prop, two prooc chains disulfide linked at the carboxy-propeptides; pray, three procu chains disulfide linked at the carboxy-propeptides; pa, proa chain missing the carboxy-propeptide; pNol-Col I, that portion of the amino-propeptide from the oil chain which is liberated by digestion with bacterial collagenase.

433 0003-2697/79/100433-15$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

434 GERARD AND MITCHELL

of a mitochondrial enzyme, P-OH butyrate dehydrogenase (1 l), and of staphylococcal a-toxin (12). The theory and applications of the procedure have been reviewed by Bock et al. (13). By adsorption of procollagen from liter quantities of serum-free human fibroblast tissue culture medium to CPG bead columns and subsequent differen- tial elution, milligram quantities of type I procollagen have been obtained in a rapidly accomplished first step. Subsequent chromatography on DEAE-cellulose to separate genetic types I and III and finally gel ex- clusion on agarose to remove partially de- graded intermediates yields a collagenase sensitive, disulfide-linked procollagen with both amino and carboxy propeptides intact.

MATERIALS AND METHODS

Tissue culture. Continuous cultures of human foreskin fibroblasts were established from primary explants of neonatal tissue by established methods (14) modified by using Ham’s medium F- 12 (15). Established cultures were grown to confluency in 150-cm* flasks (Coming) in 30 ml of complete medium defined as minimal essential medium (GIBCO, Cat. No. F-11) which contained 2.2 g/liter NaHCO,, 10% heat-inactivated fetal calf serum (KC Biologicals Inc., Cat. No. 3000), 20 mM Hepes (Sigma, Cat. No. H-3375), and 50 (uglml gentamycin (Sigma, Cat. No. G-3632). Confluent cultures were subculti- vated with a solution of 0.05% trypsin (Sigma, Type III, Cat. No. T-8253) in Puck’s saline using a 1:8 split ratio. For procollagen pro- duction, cells were planted and grown to confluency in 55 ml complete medium in roller bottles with 1200 cm2 of surface area which had been pretreated with magnesium acetate (16). Confluent cultures were between 9 and 20 cumulative doublings in age. At confluency, roller cultures were rinsed twice with 25 ml Dulbecco’s phosphate- buffered saline without CaCl, (PBS) (GIBCO, Cat. No. K-13) and preincubated 24 h in 55 ml serum-free minimal essential medium

which contained 2.2 g/liter NaHCO,, 50 U/ml penicillin, and 50 &ml each of streptomycin, ascorbate (Sigma, Cat. No. A-7506) and ,&aminoproprionitrile fumarate (Sigma, Cat. No. A-3134). Incubations were then performed for 24 h using 55 ml of the same medium as used in the preincubation; at times, this also included the addition of 1 @i/ml L-[5-3H]proline (Schwartz/Mann, Cat. No. 034142). Culture media was har- vested, chilled on ice, adjusted to 25 mM EDTA, centrifuged at 5,OOOg at 4°C for 30 min to remove cell debris, and frozen at -20°C for storage. Up to five successive 24-h incubations were performed with the roller cultures after which they were allowed to remain for 3-4 days in complete medium in which penicillin and streptomycin were substituted for gentamycin. Roller cultures were used for up to three series of 5-day incubation periods.

Risk of mycoplasma contamination was minimized by: (i) using heat-inactivated serum; (ii) the use of gentamycin when possible; (iii) heat treating trypsin solutions at 90°C for 15 min at pH 4.0 prior to use (17); (iv) keeping continuous culture periods as brief as possible by maintaining a large supply of aliquoted cell stocks frozen at -70°C or in liquid nitrogen which were thawed, expanded for roller culture, and used for incubation only as needed. Taking these precautions, no mycoplasma could be detected in cultures either by autoradiog- raphy of the cell layer using [3H]thymidine or by growth from used culture medium on agar plates specifically prepared for mycoplasma growth.

Procollagen isolation by adsorption to CPG. All preparative procedures were performed at 4°C unless otherwise speci- fied, The CPG (Electra Nucleonics Inc., Fairfield, N. J.) used was CPG-10, 120/200 mesh with a nominal pore diameter of 350 A and surface area of about 60 m”/g. A CPG column was poured of bed volume equal to 10% of the incubation volume to be applied and was equilibrated in PBS containing

PROCOLLAGEN ADSORPTION TO CONTROLLED-PORE GLASS BEADS 435

25 mM EDTA (PBS-EDTA). Up to 5 liters of serum-free culture medium was thawed and applied by gravity flow to the CPG column. The column was washed with 2-3 column volumes of PBS-EDTA after which noncollagenous proteins were eluted with 3 column volumes of 1 M potassium phosphate (Fisher), pH 8.2, which contained 25 mM EDTA. The column was again washed with 2 column volumes of PBS-EDTA. Procollagen was then eluted with 3 column volumes of 1 M Tris (Sigma), pH 8.2, measured at 4°C containing 25 mM EDTA. Used CPG was regenerated as previously described (13).

DEAE-cellulose chromatography. The procedure used to separate type I from type III procollagen was a modification of that used by Smith et al. (18). A stock solution of 8 M urea (Aldrich, Cat. No. U270-9) was prepared and deionized on a mixed bed resin (Rexyn I-300, Fisher, Cat. No. R-208) to a conductivity of l-2 pmho immediately before use. The stock urea solution was used to make the buffer for chromatography which contained 2 M urea, 50 mM Tris, pH 8.2, at 4”C, and 2.5 mM EDTA. The procollagen fractions of the CPG 1 M Tris eluate were pooled and dialyzed against this buffer with sufficient volume and changes over 36 h to effect equilibration to within 0.01% of the content of the dialysis buffer. Either a lo- or a SO-ml bed of DEAE-cellulose (DE-52, Whatman Ltd.), equilibrated in the above buffer, was poured in a 1.5 x 30-cm or a 3.5 x 30-cm column, respectively. Pro- collagen in the same buffer was applied at 4°C and the column was washed with 5 vol of buffer. Elution of procollagen was achieved using a linear gradient of O-O.2 M NaCl in the equilibrating buffer of either 200 ml for the smaller column or 800 ml for the larger column. Type I procollagen fractions were pooled and dialyzed exhaustively against 0.5 M NaCl, 50 mM Tris, pH 8.2, 2.5 mM EDTA, at 4°C. Type I procollagen was stored at 4°C in this buffer for up to several weeks.

Molecular sieve chromatography in 1 M CaCl,. A modification of the procedure of Piez (19) was employed. Running buffer, which contained 1 M CaClz (Fisher), 50 mM Tris, pH 7.6, was vacuum filtered through Whatman No. 44 paper. Bio-Gel A-5m 200-400 mesh (Bio-Rad) was equilibrated in the buffer and a 440-ml bed poured at 23°C using a 2.5 x loo-cm column. Type I procollagen was dialyzed at 4°C against the calcium-containing buffer. The sample was then concentrated to a volume of 1.5-2.5 ml either by dialysis at 4°C against buffer, made 30% in polyethylene glycoi (PEG-20) (Aqua- tide III, Calbiochem), or by dry dialysis at 4°C with Sephadex G-200 (Pharmacia). The concentrated type I procollagen was then further dialyzed against buffer at 4°C overnight. The dialyzed concentrated procollagen sample was heated to 45°C for 30 min to assure maximal denaturation of the collagen fold. Any precipitation present in the sample was removed by centrifugation at 4,000g for 20 min, and the supernatant was then applied to the column. Flow was maintained at 7.5 ml/h at 23°C using a constant pressure head of 45 cm, and 2.4-m] fractions were collected. Appropriate fractions were pooled, concentrated at 4°C by Sephadex dry dialysis or dialysis against 30% PEG-20, dialyzed against the appropriate buffer, and used for analysis.

SDSlagaroselpolyacrylamide gel electrophoresis. Due to the poor penetration of collagen chains into conventional polyacrylamide gels, a modification of the composite agarose-acrylamide gel system of Peacock and Dingman (20) was developed for electrophoresis of procollagen species in the presence of SDS. Similar systems have been used for analysis of cross-linked rabbit skeletal muscle myosin (21) and em- bryonic chick procollagen (22). Gels containing 2% acrylamide and 1% agarose were prepared as follows. A 2% agarose solution was prepared by refluxing 0.45 g agarose (Bio-Rad, Cat. No. 162-0100) in 22.5 ml deionized water in a boiling water bath for


15 min, followed by cooling to 47°C. A 5x solution of electrophoresis buffer (5E) was prepared which contained 0.2 M Tris, pH 7.2, 0.16 M sodium acetate, 5 mM EDTA, and 0.5% SDS (Bio-Rad). The stock acrylamide solution contained 20% acrylamide (Ultrapure, Polysciences, Cat. No. 0019) and 1.5% diallyltartaramide (Bio-Rad). A solution containing 9 ml of 5E, 4.5 ml of stock acrylamide, and 8.1 ml of deionized water was heated to 47°C. The agarose and acrylamide solutions were mixed and 0.45 ml each of 10% TEMED and freshly made 10% ammonium persulfate were added. The gel solution was withdrawn into a 60-ml syringe and gels were rapidly poured from the same syringe, to assure reproducibility, into 0.6 x 1 l.O-cm glass tubes against a flat paratilm bottom. Gels were allowed to polymerize overnight at room temperature. To prepare gels for running, each tube was inverted so that the flat end became the gel top. The gel was allowed to slide down the tube leaving 9.5 cm of length inside the tube cutting off the excess gel with a scalpel to leave a flat bottom end. A cheesecloth square was affixed to the end of the tube to prevent the gel from sliding out of the tube during the run. The electrophoresis buffer and the gel contained 40 mM Tris, pH 7.2, 33 mM sodium acetate, 1 mM EDTA, and 0.1% SDS. Samples for electrophoresis were concentrated either by dialysis against 30% PEG-20 or by dry dialysis with Sephadex G-200. Concentrated samples were dialyzed against a IO-fold dilution of electrophoresis buffer which contained 10% glycerol, 1% SDS, and 0.0025% phenol red. Prior to application, 50-h aliquots of samples with or without 2.5 A 0.5 M dithiothreitol (DTT) were boiled 5 min in a 100°C water bath. Samples were applied to gels and electrophoresis was performed at 1.5 mA/gel for 25 min and con- tinued at 3-5 mA/gel for 5-6 h. Gels were stained for 2 h in 0.1% Coomassie R-250 (Bio-Rad) in 25% methanol, 10% acetic acid and then destained in a stirring destainer in the same solvent. Destained gels were

photographed using a bandpass optical filter (Baird-Atomic) with a central wavelength of 554 nm. Even though gels contained only 2% acrylamide, the presence of 1% agarose afforded excellent mechanical stability and rigidity to an extent which even precluded any appreciable change in gel length upon staining and destaining. As such, mobilities of various bands from gel to gel could be directly compared by careful alignment of the tops of gels. The validity of such comparison was verified by gel runs in which identical standards run on duplicate gels were observed to comigrate as judged by the above method. When different quantities of standard proteins were run on duplicate gels, the band centers were still observed to correspond, even though larger quantities of applied protein yielded wider bands. In some cases, destained gels were sliced and solubilized with 2% periodic acid at 87°C for scintillation counting. Re- coveries of dpm generally ranged between 60 and 70%.

Electrophoresis in SDS on polyacrylamide gels (SDS-PAGE) of the collagenase digest of type I proy was performed in 10% polyacrylamide gels using the Tris-acetate buffer system as described. Gels were poured at room temperature using a solution which contained 15 ml 20% acrylamide, 0.53% bisacrylamide (Polysciences), 6 ml 5E, 8.7 ml deionized water, and 0.15 ml each of 10% TEMED and 10% ammonium persulfate. Gels were overlayered with 0.1% SDS, 0.05% TEMED, and 0.05% ammonium persulfate and allowed to polymerize for 3 h. Sample preparation, electrophoresis conditions, staining, and photographic procedures were essentially identical to that described for the agarose-acrylamide system. For radioactivity profiles, either stained or unstained gels taken immediately after electrophoresis were sliced in l-mm sections and solubihzed for scintihation counting in 0.4 ml 30% hydrogen peroxide at 85°C for 6 h.

Collagenase digestion. Collagenous


sequences in isolated human fibroblast culture proteins were demonstrated by incubation with purified bacterial collagenase (5275 CLSPA, Worthington Biochemical Corp.). Incubations were performed in the presence of TLCK to inhibit any con- taminating Clostripain activity (23). No proteolytic activity could be detected when several noncollagenous proteins were incubated under these conditions (data not shown). Incubations contained 50 pg of either lathrytic rat skin collagen or human procollagen with or without 0.5 pg collagenase in 1 ml of 0.5 M NaCl, 50 mM Tris, pH 7.3, at 37”C, 5 mM CaCl,, and 3.4 x lops M TLCK. Samples were incubated at 37°C for 90 min. The digestion was terminated by the addition of 0.1 ml 10% SDS and heat- ing in a 100°C water bath for 2 min. Samples were dialyzed in Spectrapor 1 dialysis tubing (Spectrum Medical Industries) at room temperature against 0.5 M NaCl, 50 mM Tris, pH 7.6,2.5 mM EDTA, and 0.1% SDS, concentrated in the same buffer made 30% in PEG-20, and then prepared for electrophoresis as described.

Scintillation counting. Aliquots of aqueous samples containing [3H]proline were counted in lo- 15 ml cocktail which contained 0.34% (w/v) BBOT (Eastman), 6.8% (w/v) naph- thalene (scintillation grade, Amersham), 51.6% (v/v) toluene (Scintanalyzed, Fisher), and 40% (v/v) ethylene glycol monomethyl ether (Fisher). Samples were counted in a Beckman model LS-233 liquid scintillation counter. All values were converted to dpm using external standard determining the standard efficiency curve for each series of counts. Counting efficiency ranged between 30 and 40%.

Protein estimation. Procollagen protein concentrations were estimated by tryptophan fluorescence using freshly prepared solutions of BSA as standard. Fluorescence was measured on an Aminco Bowman spec- trophotofluorimeter using A excitation of 278 nm and X emission of 340 nm. Factors which encouraged the use of this method

include high sensitivity (l- 10 &ml), complete recovery of the sample, amenability to all of the buffer systems employed in this purification procedure, and convenience in monitoring column effluents. Subsequent to the accumulation of larger quantities of purified procollagen, estimation of pro-y(I) concentration by tryptophan fluorescence was compared to estimation by the method of Lowry et al. (24). Since rat skin collagen was observed to be 20-30% less reactive than BSA in the Lowry assay, a composite standard curve was derived based on an approximation for procollagen of two-thirds collagenous sequences and one-third noncollagenous sequences. The estimates of pray(1) concentration obtained by tryptophan fluorescence were thereby found to agree with the Lowry estimates within a 25% margin of error. However, the tryptophan content of human procollagen and its various processed forms studied in this laboratory has not yet been determined. Further, even though BSA standards were always prepared in the same buffer of the procollagen being measured, the quantita- tive effect of ionic environment on BSA tryptophan fluorescence cannot necessarily be assumed to correlate to procollagen tryptophan fluorescence. Therefore, all estimates of procollagen concentration reported in this investigation as measured by tryptophan fluorescence must be regarded as an approximation.

RESULTS

Procollagen is efficiently adsorbed from tissue culture medium by columns of CPG beads. As long as the bed volume of CPG was at least 10% of the applied 24-h serum- free tissue culture medium volume, no collagenous material could be recovered by 20% ammonium sulfate precipitation of the CPG pass through. Following adsorption on CPG and a 1 M phosphate wash, procollagen and a variety of degradation products were eluted with 1 M Tris as illus-


trated in gel 1 of Fig. 1. The predominant form was undegraded type I procollagen [pray(I)]. In addition, proy(II1) was present as well as some type I partially processed intermediates. No partially processed type III procollagen forms could be identified by SDS-agarose-acrylamide electrophoresis of human fibroblast culture proteins isolated from the culture medium. This latter finding is in agreement with the results of Goldberg (25). Of the total material recovered by elution of CPG with 1 M Tris and 1 M phosphate, averages of 88% of the total dpm of [3H]proline and 36% of the total tryptophan were recovered in the Tris fraction. Routinely, an average of 4 pg of human procollagen per milliliter of culture medium could be recovered from the CPG by elution with 1 M Tris, 90% of the total eluting between 70 and 120% of the CPG column volume. The efficiency of elution of procol-

lagen from CPG by 1 M Tris was demonstrated by subjecting CPG stripped with Tris to 1% SDS at room temperature. Ap- proximately 14% additional material was eluted relative to the Tris elution. SDS- agarose-acrylamide electrophoresis of this material revealed primarily lower- molecular-weight proteins and an absence of proy forms. If the phosphate wash step was eliminated, analysis of the Tris eluate by SDS-agarose-acrylamide electrophoresis (not shown) revealed: (i) the presence of a disulfide-linked band of mobility intermediate to the proy band and the y band of rat skin collagen, (ii) diminished relative presence of the type I proy band as judged by Coomassie blue staining, and (iii) the presence of a disulfide-linked lower-molecular-weight band of mobility corresponding to approximately 100,000. Alternatively, if a 1 M Tris elution was performed without

I 2 3 4 5 6 7 8 9 IO II 12

PRO7ulr)

PROXCI) -

PROP - l

PROdI -

PROMI red

PROa2& f

J MYOSIN

d-g -MYOSlN

b--a

TRIMER

DIMER

MONOMER

FIG. 1. SDS-agarose-acrylamide electrophoresis of procollagen fractions. Sample preparation and electrophoresis was performed as described. Samples for gels 3-10 were taken from Bio-Gel A-5m chromatography in 1 M CaCI,. (Gel 1) CPG 1 M Tris-eluted procollagen; (Gel 2) DEAE-cellulose-purified type I procollagen; (Gel 3) type I pray; (Gel 4) type I pray reduced; (Gel 5) prop; (Gel 6) prop reduced; (Gel 7) proal; (Gel 8) proal reduced; (Gel 9) pu; (Gel 10) pa reduced; (Gel 11) rat skin collagen; (Gel 12) rabbit skeletal muscle myosin.


a prephosphate wash and 1 mM phenyl- methylsulfonyl fluoride and 10 mM N-ethyl- maleimide were included in all buffers, an agarose-acrylamide electrophoretogram of this material (not shown) closely resem- bled that of the 1 M Tris eluate from phosphate-washed CPG beads (Fig. 1, gel 1). Moreover, incubation of type I procollagen with aliquots of the phosphate wash resulted in the generation of the intermediate proy band in SDS-agarose-acrylamide electrophoretograms (not shown). Thus, it appears that the phosphate wash prior to Tris elution effectively removed one or more proteases which degrade procollagen. The stability of proy as judged by the absence of intermediate proy generation in subsequent purification steps is confirmatory evidence for this conclusion. Examination of the phosphate-eluted material by SDS- agarose-acrylamide electrophoresis (not shown) revealed primarily lower-molecular-

weight proteins and a lack of the procollagen forms observed in the Tris eluate (Fig. 1, gel I).

Resolution of type I from type III procollagen was accomplished by chromatography of the CPG Tris eluate on DEAE- cellulose. A representative elution profile is shown in Fig. 2. Only a small amount of material eluted in the column pass through which represented primarily collagen chains as judged by comigration on SDS-agarose- acrylamide gels with the ml and (~2 chains of rat skin collagen (not shown). The material which eluted in the larger peak between fractions 42 and 56 and in the smaller peak between fractions 60 and 72 represents type I and type III procollagens, respectively, as previously reported (5,18). The electrophoretic profile of the type I procollagen (Fig. 1, gel 2) demonstrates the presence of a predominant proy band in addition to prop, prool, and pa forms (to be subse-

GRADIENT

THROUGH

FRACTION

FIG. 2. DEAE-cellulose chromatography of CPG 1 M Tris-eluted procollagen. The 880 ml of incubate, which included 1 &i/ml [3H]proline, was adsorbed and eked from a lOO-ml CPG column as described. The 1 M Tris eluate, containing approximately 1.5 mg procollagen proteins, was dialyzed against the described buffer and applied to a 1.5 x 30-cm column of DEAE-cellulose (lo-ml bed equilibrated in the same buffer). Elution was achieved with a 200-ml gradient of O-O.2 M NaCl in the same buffer and 2.5 ml fractions were collected. (-B-) Tryptophan, (-0-) dpm of 13H)pro1ine.


quently discussed). When the type III pro- acrylamide electrophoretic profiles of the collagen was analyzed in the same gel sys- leading and trailing edges of the type I pro- tern (not shown), primarily one band was collagen peak suggesting that the double observed under nonreducing conditions type I peak is an elution artifact. Neverthe- which migrated behind the type I proy band. less, successful removal of the type III pro- Under reducing conditions, this band was collagen from the Tris CPG eluate was replaced by a single band migrating just achieved in this second phase of purifica- behind the reduced proal band. tion. Yields of total material applied ranged

Type I procollagen often eluted as an between 75 and 85% by tryptophan fluores- apparent double peak on DEAE-cellulose cence and dpm of [3H]proline. as shown in Fig. 2, or with a broadened Final purification of human type I proy trailing shoulder. Attempts to resolve two was achieved by molecular sieve chroma- peaks by elution with a shallower gradient tography at room temperature on Bio-Gel produced a type I procollagen peak with a A-5m in 1 M CaCl,, 50 mrvt Tris, pH 7.6. further broadened trailing edge. In addition, A representative elution profile is illustrated no significant differences could be detected in Fig. 3. A small amount of material was between the Coomassie blue SDS-agarose- seen to elute in the void volume and pre-

PROY

1

Pa

PHENOL RED

60 80 100 120 IYO 160 180 FRACTION

FIG. 3. Molecular sieve chromatography of type I procollagen on Bio-Gel. A-Sm in 1 M CaCl,. DEAE-cellulose-purified type I procollagen containing approximately 1 mg protein was isolated as described from incubation medium containing [3H]proline. Type I procollagen was dialyzed against 1 M CaCl, buffer at room temperature, concentrated to 1.6 ml, and then dialyzed again against the same buffer. The sample was heated to 45°C for 30 min prior to application to a 440 ml B&Gel A-5m column, 2.5 x 100 cm, equilibrated in the described buffer. Flow rate was maintained at 7.5 ml/h, and 2.4-m] fractions were collected. (-S) Tryptophan, (-0-) dpm VH]proline.


sumably represents very high-molecular- weight aggregates. The major peak to elute was identified as proy. Electrophoretograms of the pooled fractions from the leading half of the peak (to fraction 80) revealed a single band under nonreducing conditions (Fig. 1, gel 3) with mobility similar to that of myosin trimer. Reduction with DTT resulted in the appearance of two bands of lower-molecular-weight (Fig. 1, gel 4). The more intensely staining and slower migrating band is the precursor for the (Y 1 collagen chain (procu 1 red) while the less intensely staining and more rapidly migrating band represents the precursor for the (~2 collagen chain (proa2,,). The procul:procu2 ratio of proy was estimated by measuring the dpm of [3H]proline in whole procul and procu2 bands excised from agarose-acrylamide gels on which proy had been electrophoresed under reducing conditions. The average ratio calculated from four determinations was 1.84 (kO.23 SD).

The second major peak to elute from the Bio-Gel column (Fig. 3) contained two pro/3 bands under nonreducing conditions (Fig. 1, gel .5), which migrated in the region of myosin dimer, suggesting a one-third reduction in mass relative to proy. Electrophoreto- grams under reducing conditions (Fig. 1, gel 6) yielded two bands, one of which comigrated with the proalred band of reduced proy (Fig. 1, gel 4). It is suspected that the other faster-migrating less intensely staining band of the reduced pro/3 gel represents a precursor to the (~2 chain. However, the mobility of this band was reproducibly observed, as shown in Fig. 1 and in previous gel runs (not shown), to be slightly greater than the procz2,ed band of reduced proy (Fig. 1, gel 4). This band may therefore represent a truncated form of the proa chain.

Procul eluted after and cleanly separated from the profi peak (Fig. 3). That this peak represents procul chains is suggested in part by the electrophoretic profile under reducing conditions (Fig. 1, gel 8) in which the

band was seen to comigrate with the reduced procvl chain of proy. Under nonreducing conditions (Fig. 1, gel 7), the band is retarded in mobility and comigrates with a weak band in the Tris eluate from CPG (Fig. 1, gel 1). Procollagen chains missing the carboxy- terminal propeptide, termed pa, eluted on the trailing edge of the procul peak (Fig. 3). Several observations support this identifica- tion. No tryptophan peak in the Bio-Gel elution was observed to correspond to this peak of dpm of [3H]proline, which suggests that the carboxy-terminal propeptide is missing (26). The electrophoretic mobility of the al component of this material under reducing conditions (Fig. 1, gel 10) was observed to be intermediate to that of the rat skin al chain (Fig. 1, gel 11) and the reduced procul chain (Fig. 1, gel 4). In addition, the change in electrophoretic mobility of this band upon reduction with DTT (Fig. 1, gels 9 and 10) is consistent with the presence of cystine disulfide bonds (27). It can be in- ferred that in the absence of the carboxy- terminal propeptide, the cysteine residues in this material correspond to those of the amino-terminal propeptide.

Some clarification of identity of gel bands seen in Fig. 1 is needed at this point. Though the differences in mobilities of some of the bands are indeed small, these differences as have been discussed were reproducible. As discussed under Materials and Methods, the alignment of gel tops allows band mobilities to be visually compared from gel to gel. Reproducibility of migration is demonstrated in the comigration of the pray(1) band in gels 1, 2, and 3 and in the more rapidly migrating bands of gels 1 and 2. One point of confusion is the apparent presence of proa!lred in gels 1 and 2 which were run under nonreducing conditions. It is believed that this band in gels 1 and 2 represents pal, as judged by comigration with the unreduced pcul band in gel 9. Further, since a pa peak was demonstrated in Bio-Gel chromagraphy (Fig. 3) of applied sample corresponding to gel 2, it is consistent to identify a pal


band in this gel. Likewise, a less intensely staining band migrating just ahead of proa2,d is observed in gels 1 and 2. For the same reasons just discussed, it is believed that this band represents a pa2 band. In this case, however, no such band is visualized in Fig. 1 in the gel of unreduced pa, gel 9. When the per sample of gel 9 was overloaded (not shown) and run under nonreducing conditions, however, a faint band was observed which comigrated with the faint band of gels 1 and 2 under discussion. The sample used for electrophoresis of the pcl! peak from Bio-Gel chromatography (Fig. 3) was fraction 109, the peak fraction by dpm. A mini- mally detectable but reproducible shoulder can be observed by the dpm elution protile of the p’~ peak in Fig. 3, corresponding to fractions 111 and 112. It is suspected that this shoulder represents a p(r2 component of the p’~ peak with the earlier eluting and more predominant portion of the peak representing pal. If this were the case, it might then account for an apparent diminished presence of a putative pa2 band in gels 9 and 10. Once again, the identity of the pa2 band on agarose-acrylamide gels is suggested by a mobility of this band under reducing conditions (not shown) intermediate to that of the a2 band of rat skin collagen and pro(Y2,,d.

In the Bio-Gel elution (Fig. 3), the apparent ratio of tryptophan to proline for the proy peak is lower than for the prop and procu peaks as judged by the tryptophan fluorescence and dpm elution profiles. If proline is taken as an index of collagen sequences and tryptophan of carboxy-terminal propeptide (26), then the procu and pro/3 forms are seen to have a higher content of carboxy-terminal propeptide per collagen chain than proy. If the agarose-acrylamide electrophoresis gels were overloaded, the reduced prop and procu revealed the presence of a low-molecular-weight component which migrated close to the end of the gel (not shown). This band was not observed for the unreduced counterparts or for the

reduced or unreduced pro-y and pa. This observation in conjunction with the apparent tryptophan to proline ratios from the Bio- Gel elution suggests that the partially processed prop and prolv forms have retained the carboxy-terminal propeptide(s) generated by limited proteolysis of the proy chains by virtue of interchain disulfide bonds.

Recoveries on the Bio-Gel column (Fig. 3) averaged 98% (k-3.5% SD) of the applied tryptophan over five runs subsequent to the first two runs. The recoveries for the first and second runs using the same criteria were 50% and 80%, respectively. It was concluded that initially, some of the procollagen was lost due to adsorption to the Bio- Gel matrix, but that subsequently, the ca- pacity to adsorb was saturated. The use of molecular sieve chromatography on Bio-Gel A-5m in 1 M CaCl, for procollagen purification therefore allows virtually a complete recovery of the applied material.

The presence of collagen sequences was demonstrated in each of the procollagen forms shown for the Tris CPG eluate (Fig. 1, gel 1) by the disappearance of these bands subsequent to collagenase digestion (not shown). Evidence for the presence of both the amino- and carboxy-terminal propeptides was obtained by collagenase digestion of pray(1) labeled with [3H]proline and analysis of the collagenase resistant peptides by SDS-polyacrylamide gel electrophoresis on 10% gels. Resolution of the propeptides using [3H]proline was made possible owing to their significant content of proline. Values of approximately 11 and 5% of the total residues per mole as proline have been reported for the collagenase-liberated proal amino- terminal propeptide (pNal-Co1 1) in calf and dermatosparactic sheep (28) and for the carboxy-terminal propeptide trimer from chick embryo tendon (29), respectively. In Fig. 4, the lower unreduced SDS gel (U) and its radioactivity profile of the collagenase digest of proy show the intact carboxy- terminal trimer at slice 11 and pNal-Co1 1 at slice 54. In the upper reduced gel (R) and


SLICE

FIG. 4. The 10% acrylamide SDS-PAGE of collagenase digest of type I proy. Type I proy was purified as described from incubation medium which contained [3H]proline. Collagenase digestion was performed and the sample was run both reduced with DTT (R) and unreduced (U). Both gels were stained with Coomassie blue, photographed, and subsequently sliced into l-mm sections and solubilized for scintillation counting. The standard proteins shown above prepared identically to the reduced sample were: phosphorylase a (P), bovine serum albumin (B), y-globulin heavy chain (H), ovalbumin (0), y-globulin light chain (L), myoglobin (M), and cytochrome c (C). The origin is on the left. (x) dpm [3H]proline of unreduced gel, (@) dpm [3H]proline of reduced gel.

its radioactivity profile of the same material more rapidly migrating material is observed shown in Fig. 4, the carboxy-terminal trimer in the region of slices S-70. The nature peak has been replaced by the free proal of this material has not been further investi- and proa carboxy-propeptides, corre- gated. Alternatively, electrophoresis of the sponding to slices 44 and 46, respectively. collagenase digested proy sample treated The slower-migrating procul carboxy-pro- with iodoacetamide with and without prior peptide appears clearly more predominant DTT reduction demonstrated an equivalent that the faster-migrating proa carboxy- pNal-Co1 1 band in both unreduced and propeptide as judged by both intensity of reduced conditions. These samples were staining and radioactivity profile. In the prepared for electrophoresis by a modifica- reduced gel, however, a distinct pNaI-Co1 1 tion of the method of Lane (30). For reduc- band as seen in the unreduced gel is not tion, samples were mixed with l/50 volume observed in either the stained gel or radio- of DTT and boiled for 2 min. Reduced and activity profile. In the radioactivity profile, unreduced samples were then treated with


l/20 volume of il.25 M iodoacetamide at 50°C for 15 min and then applied to gels. Under these conditions, however, resolution was decreased to an extent that precluded visualization of separate reduced procr 1 and procu2 carboxy-propeptide bands.

The ratio of total dpm in the carboxy- terminal trimer peak to the total dpm in the pNal-Co1 1 peak is in very good agreement with a ratio calculated from the reported proline content of these propeptides. From the values of 48 residues of proline per mole of carboxy-terminal propeptide trimer from chick embryo tendon (29) and 10 residues of proline per mole of pNal-Co1 1 from sheep (28), a molar ratio of 2.4:1 can be derived for procollagen (digestion of 1 mol of procollagen yields 1 mol of carboxy-terminal propeptide trimer and 2 mol of pNal-Co1 1). From Fig. 4, the ratio of radioactivity in slices 9-14, corresponding to carboxy- terminal propeptide trimer, to the radioactivity in slices 52-57, representing pNal- Co1 1, is equal to 2.5. A separate gel run of a different collagenase digestion of proy (not shown) yielded virtually the same ratio for the same two peaks. The agreement of the observed ratio with that predicted from the reported proline composition of the carboxy-propeptide trimer and of pNal-Co1 1 from other species is consistent with the presence of these propeptides in human type I procollagen.

Molecular weights for the procollagen propeptides were estimated by comparison of mobilities by SDS-PAGE to those of the standard proteins shown in Fig. 4. Values calculated and averaged from two runs were 34,900 for the procul carboxy-propeptide, 32,000 for the procu2 carboxy-propeptide, and 26,500 for the pNal-Co1 1 peptide. Even though molecular-weight determinations by SDS-polyacrylamide electrophoresis for both the amino- (28) and carboxy- (31) propeptides have been reported to be anomolous , the values calculated are in good agreement with those values as determined by SDS- PAGE previously reported (28,29,31). These

results are consistent with extension peptides at both the amino- and carboxy- termini of the tropocollagen region of proy (I) with interchain disulfide bonds present between carboxy-terminal propeptides.

DISCUSSION

A new technique for isolation of human type I procollagen from fibroblast culture media has been described which eliminates procollagen-directed proteolytic activity among other noncollagenous proteins at the initial stage of isolation. The type I proy so isolated is subsequently purified with minimal risk of further modification by en- dogenous procollagen peptidase(s) which might otherwise generate partially processed intermediates in the course of purification. In the same CPG isolation procedure, a 20-fold concentration of procollagen from the culture medium is effected, and medium components, including free E3H]proline if present, are eliminated in the pass-through volume. The CPG adsorption procedure is rapid in that processing of 5 liters of medium required only 7 h from the start of application to the retrieval of procollagen-containing fractions of the 1 M Tris eluate. In addition, the glass matrix is readily regenerated for subsequent reuse indefinitely.

The nature of the proteolytic activity removed in the 1 M phosphate elution has not been characterized. However, it was observed that if a 1 M Tris elution of the CPG column was performed without a prephosphate elution, a disulfide-bonded lower- molecular-weight band of about 100,000 was seen by SDS-agarose-acrylamide electrophoresis (not shown). It is unclear as to whether this represents a type I- or a type III-derived product. Also under these conditions, agarose-acrylamide electrophoresis revealed that a disulfide-bonded py band was generated at the expense of pro-y(I) of mobility intermediate to the type I proy band and the y band of rat skin collagen (not shown). When material from the phos-


phate eluate was mixed with type I procollagen, agarose-acrylamide electrophoresis revealed the appearance of this same intermediate py band (not shown). This evidence suggests that procollagen peptidase(s) specific for type I procollagen, possibly in conjunction with other proteases, are isolated in the 1 M phosphate eluate, although no definitive evidence has been obtained.

The presence of partially processed type I intermediates in the 1 M Tris eluate, as seen in gel 1 of Fig. 1, alludes to proteolytic modification of procollagen isolated by this methodology. Some of this proteolysis may be attributable to processing in the culture medium during the 24-h incubation period. However, the relationship of culture incubation time to the relative distribution of pro?(I) and processed forms was not investigated. Alternatively, the possibility that some proteolysis has occurred during the CPG isolation procedure cannot be ruled out. EDTA has been reported to inhibit procollagen peptidase activity isolated from human fibroblast culture medium (32) and from extrac- tions of normal and pathological human skin biopsies (33). Accordingly, 25 mM EDTA was included in all solutions employed in the isolation procedure to minimize any further modification of human procollagen by procollagen peptidase(s) present in the culture medium. Nonetheless, initial fractionation of culture proteins on CPG was effective in removing peptidase activity specific for type I procollagen as discussed above. In particular, expression of the activity isolated in the phosphate eluate re- sponsible for the generation of a py form would preclude final purification of pray(1) by molecular sieve chromatography.

Subsequent to the fractionation of type I procollagen from type III on DEAE-cellulose, human type I proy was purified to relative homogeneity in 1 M CaCl, on Bio-Gel A-5m. Two observations suggested that in the pro/3 and proa forms, carboxy-propeptides cleaved from their collagen chains were still covalently attached via disulhde

associations within the carboxy-propeptide trimer. First, the tryptophan:proline ratio for the proy peak was lower than for the prop and proa peaks in the Bio-Gel elution, as judged by dpm and tryptophan fluorescence elution profiles (Fig. 3). In addition, reduction of overloaded prop and proa samples generated a lower-molecular-weight band observed by agarose-acrylamide electrophoresis which migrated near the end of the gel (not shown). This band was not observed for reduced proy under the same conditions. These results are consistent with those of Davidson et al. who have reported increasing tryptophan:proline ratios of successive intermediates of chick bone procollagen on SDS-PAGE (34), and have further proposed a model for the conversion of procollagen to collagen in the same system in which successive intermediates covalently retain cleaved carboxy- propeptides via disulfide bonding to the uncleaved carboxy-propeptide (35). In con- trast to the results of Davidson et al., however, our results suggest the presence of pa forms of human procollagen containing amino-terminal but devoid of carboxy-terminal propeptides, although definitive evidence has not yet been obtained.

Examination on SDS-PAGE of peptides liberated from pray(1) following bacterial collagenase digestion indicated the presence of proal and proa carboxy-terminal and procul amino-terminal propeptides. The proa amino-terminal propeptide of dermatosparactic sheep has been shown to be cleaved into small peptides by bacterial collagenase (36), presumably due to the presence of collagen sequences included within the propeptide domain. If a similar structure were to exist for the human proa peptide, bacterial collagenase digestion of human type I proy might preclude its identi- fication by the procedures employed here. Moreover, the amino-terminal procollagen peptidase from calf tendon has been shown to excise the amino-terminal propeptides endoproteolytically in bloc (37). Assuming


an analogous mechanism for the human can be effectively isolated by adsorption enzyme, the indicated presence of the proclll to and elution from CPG beads (39). Al- amino-terminal propeptide on human proy though no direct data is presented, it seems suggests a lack of processing by this enzyme likely that this method will have applicability and, thus, the presence of the procr2 amino- to other genetic types of procollagen as well terminal propeptide. It should be emphasized as the isolation of procollagen peptidases. that the evidence presented here for the presence of amino- and carboxy-terminal ACKNOWLEDGMENTS propeptides in human pray(1) is circum- stantial and is based in part on the charac-

We are grateful to Dr. Dixie Frederiksen and Dr.

terization of these propeptides from non- William F. Harrington for their generous contributions of rabbit skeletal muscle myosin. Thanks also go to

human sources. However, these results are Mr. John Sonneborn and Mrs. Shirley Schuffman for consistent with those of Lichtenstein et al. their technical assistance and expertise. Supported

(38) who demonstrated by mammalian col- in part by NIH Grams AM 18222 and T 32 GM 073t9.

lagenase digestion the presence of both amino- and carboxy-terminal extension peptides on human fibroblast type I procollagen.

A key feature of the three-step isolation and purification procedure outlined here is that precipitation of procollagen from solution is avoided. It has been our experience that a previously employed isolation method of precipitation of procollagen by ammonium sulfate always yielded some material which could not be resolubilized. When this material was examined on SDS-agarose- acrylamide gels, a predominance of higher- molecular-weight aggregates was observed. Inadvertent precipitation which could not be resolubilized was also observed in the course of various preparative manipulations, especially following concentration of procollagen solutions. Such losses coupled with adsorptive losses were sometimes significant. Concentration by dry dialysis with Sephadex G-200 though time-consuming, proved to be the most successful in mini- mizing this problem and usually afforded recoveries of 50-75%. Concentration of procollagen solutions by dilution and reap- plication to CPG is possible although this has not been adequately investigated.

Adsorption to CPG beads has been demonstrated to be useful for the isolation, concentration, and initial fractionation of human procollagen. It has been recently demonstrated that procollagen from Chinese hamster lung cell tissue culture medium

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Documents

Isolation and purification of human type I procollagen by adsorption to glass beads