4h,\,\

I,( \I

HIO( HI hllSIRY

91.

183-188

11979)

Analysis of Collagen Cyanogen Bromide Peptides Using Electrophoresis in Continuous Concave Gradient Polyacrylamide Gels G. COLT’

WILLIAM

AND D~BOKAH

Received

June

cyanogen bromide peptidts obtained in sodium dodecyl sulfate-concave

was

recoveries

evaluated.

Improved

were

also

of small

reprint

from

cuitahle

for

the

study

collagen marker) into factor semiquantitative in insoluble dermis of some

aspects

requests used: SDS.

ethylenediaminetetraacetate.

should sodium

be arddressed. dodecyl sulfate:

human polyacrylamide

(10.000-20.000

collagen peptides 3. 6. and 8. which migrate gels, were resolved into two bands on gradient to convert the densitometry areas of the a2 CB3.5

Following the discovery in I971 that human dermis contained both type I and III coiiagens. the relative amounts of these coiiagens have been determined in the dermis obtained from normal humans and from those with connective tissue diseases( l-4). However, such studies are complicated by the insoluble nature of human collagen and the limited amount of tissue usually avaiiable for study. To some extent these problems have been overcome by cyanogen bromide cleavage of small amounts of insoiubie collagen into soluble peptides.. followed by peptide analysis using sodium dodecyi sulfate (SDS)-poiyacryiamide’ gel eiectrophoresis and Coomassie blue staiining (5). One of the disadvantages encountered with this system is the incomplete recovery of the smaller peptides (12.000-20.000 MW) when electrophoresis is carried out in gels of uniform poiyacryiamide concentration (6). With a view toward improving the recov’ To whom ” Abbreviations

peptides

III

and the ~ul(lII) CB8 (type III collagens. Using this calibration tions of type I and III collagen?

EDTA.

5. 1978

A method of analyzing collagens using electrophoresis and the type polyacrylamide was derived

A. BEAN

MW)

type

I and III gel slabs

were

obtained

as a single band gels. A calibration (type I collagen

on

7.5’; factor marker)

approximate weight ratios of these estimations of the relative proporand bone were made. Gradient gels

of peptide

involvement

in cross-linkages.

eries of the smaller peptides we have evaiuated a method of analyzing CNBr peptides of collagen using eiectrophoresis in continuous concave gradient poiyacryiamide gels. We describe the patterns obtained with peptides of human type I collagen and its component aI(I) and ~2 chains and type III coiiagen and show that improved recoveries and resolution of the smaller peptides were obtained. We also show that this method is suitable for the analysis of small amounts of insoluble collagen from dermis and bone in which a qualitative and semiquantitative assessment of the collagen types could be made as well as an assessmentof some aspects of peptide involvement in cross-iinkages. MATERIALS

AND METHODS

Rvccgc~trts. Sodium dodecyi sulfate and bromophenoi blue were obtained from B. I). H. Chemicals Limited, Poole, England. Coomassie brilliant blue R-250 was purchased from Bio-Rad L,aboratories, Richmond, California. and all other reagents

184

COLE AND BEAN

were of analytical grade and were supplied by Ajax Chemicals Limited, Melbourne.

maximum capacity of 30 ~1. The apparatus was filled with 0.05 hi sodium phosphate Pwprrrrrticvt c?f’ collrrgct~ CNBr pc~pticlr~s. buffer, pH 7.0. containing 0. l’i SDS. EquilPurified type I collagen and its component ibration of the gel with this buffer was CUI(1) and ~y2chains as well as type III col- achieved by pre-electrophoresis at room lagen were prepared from human dermis us- temperature for 1 h at 200 V. ing previously described methods (7). CySample pwprrrrrtiott (ml rtpplic~cttirttr. CNBr anogen bromide cleavage of 3-mg amounts peptides were dissolved ( 1 mgiml) in 0.01 M of these collagens was achieved using the sodium phosphate buffer, pH 7.0, containmethod described by Scott and Veis (8.9). ing 1% SDS. IO”: sucrose, and 0.1% bromoThe reaction was terminated by IO-fold dilu- phenol blue. After denaturation at 50°C for tion with deionised water followed by 20 min. 20-p] portions were introduced into freeze-drying. The peptides extracted from each sample slot. the dried material with 0.1 M acetic acid Elrc.trop1lorr.si.s. Electrophoresis was conwere freeze-dried and analyzed by electro- ducted at room temperature for 4 h at 75 V. phoresis. The buffer circulating pump was started afNoncollagenous proteins were removed ter the bromophenol blue had entered the gel. Grl strrinittg rrtzd tic~strtitting. The gels from finely sliced pieces of normal human dermis by gentle stirring in 8 M urea at 4°C were stained overnight at room temperature for 24 h (10). Human cortical bone samples with 200 ml of freshly prepared 0.25% (w/v) were decalcified with 0.4 M EDTA. pH 7.6, Coomassie brilliant blue R-250 in 505%(v/v) at 4°C. The skin and bone residues were methanol and 7% (v/v) acetic acid. The gels washed with deionized water and milled at were destained in repeated changes of a liquid-N, temperature in a freezer-mill (Spex solution containing 10% methanol and 5% Industries, Metuchen, N. J.). The powdered acetic acid. specimens were freeze-dried and 2- to 5-mg Grl .sc~ctttnitz,q. The gels were scanned at amounts were digested with CNBr using the 520 nm with a scanning microdensitometer method described above. The 0.1 M acetic (Quick Scan, Helena Laboratories, Beauacid-soluble peptides were freeze-dried and mont. Tex.). Each scan was quantitated by analyzed by electrophoresis. extending the baseline from the protein-free Elcc,tropltorclsis rcpprrrrrttrs. The vertical regions of the gel. Perpendiculars were then gel slab electrophoresis system described drawn from the midpoint of the valleys beby Margolis and Kenrick (I 1) was used. It tween adjacent peaks to the baseline. The consists of an upper gel holding portion peak outlines were traced onto paper, cut which rests on a lower divided tank con- out. and then weighed on a microanalytical taining a pump to circulate buffer through balance. a heat exchange bag. This apparatus is marketed as the Gradipore electrophoresis sysRESULTS tem by Heatherton Laboratories. MelC‘otltiitiotis of E;lrc,troplictrcJsi.s bourne. Grls. From the same company we purThe preelectrophoresis and electrophorechased polyacrylamide gel slabs (82 x 82 sis voltages used were selected according x 6 mm) cast between disposable glass to the manufacturers’ guidelines. Optimal plates. These gels showed a concave poly- resolution with reliable quantitation of miacrylamide concentration gradient from 4 nor components was obtained when 20-pg peptide loads were electrophoresed at 75 V to 26’Z. Prrclrc.trophorr.si.\. A ICslot samplespacer for 4 h. was inserted between the glass plates at the Varying staining conditions were tested 45%end of the gel slab. Each slot had a including overnight staining at room tem-

ANALYSIS

OF

COLLAGE:N

perature; 2 h at 40°C and 4 h at 40°C. As the overnight method gave the most uniform staining it was used throughout thlis study. C‘NBr

Prptidcs

,fLor?l Plrrijic)cl

C’ollrr~yc~rls

Characteristic patterns of type 1 collagen. &(I) and cu2chain CNBr peptides were ob-

PEPTIDES

185

served (Fig. 1). The peptide migration pattern was the same as we observed in 7.5% cylinder gels and the identity of each peak was determined from the relative mobility data reported by Scott and Veis (8). Both the slow and the fast migrating bands were sharp although occasionally minor trailing of the tu2 CB3.5 was observed. The recov-

1.0I (a)

TYPE I

03I

MIGRATION

FIG. (c) a2 mined

I. Electrophoretograms chains; (d) by comparison

type

of CNBr III with

peptides

(IO-20

collagen; and I:e) reduced the patterns obtained on

type 7.5%

(mm)

pg)

obtained

III collagen. polyacrylamide

from

(a) type

The identity gels (8).

1 collagen: of each

(b) al(I) peak

was

chains; deter-

186

COLE

AND

eries of small peptides, LYICB6 and tuI CB3, as determined by comparison of their densitometry areas with the area of the larger ~2 CB3.5 peptide, were close to IOOC: of the expected values (6.8). The large type 111CNBr peptides showed a typical pattern (10) and. after reduction of disulfide bonds with /$mercaptoethanol. the characteristic increased migration of the cuI(III) CB9 was observed (Fig. I). However, in gradient gel slabs two peaks were observed beyond the cuI(III) CB4, whereas only one peak, which contained the type III collagen peptides 3. 6. and 8, was observed in gels of uniform concentration (IO). When the type III peptides were extracted from this region of a 7.5% cylinder gel with 0.1 M acetic acid they were separated into two bands by electrophoresis on our gradient gels. From the molecular weight data provided by Chung et trl. (12) it is likely that the first band contained the cuI(II1) CB8 (MW 12.000)and the second band, the ~uI(I11) CB3 (MW 9500) and the aI(II1) CB6 (MW

BEAN

(SEM). culated tometry to type

A calibration factor of I. I7 was calto enable conversion of the densiarea ratios to weight ratios of type I III collagens.

A typical pattern of dermal peptides (9, 10) was obtained in which the type I and type III marker peptides were well resolved (Fig. 2). Using the calibration factor derived from the soluble collagen CNBr peptides the approximate percentage of type III collagen in this sample was calculated to be 28C’c. The dermis showed a marked reduction or an absence of the carboxy-terminal ~uI(I11) CB9. a reduction in the peak tentatively nominated as containing the amino-terminal crI(I11) CB3, and a reduced amount of the carboxy-terminal (~1 CB6. These peptides are known to take part in intermolecular cross-linkages and the yield of these peptides appears to depend on the susceptibility of these linkages to acid cleavage (9.10). 8000). The u1 CB6 obtained from the insoluble derThe (~2CB3.5 (MW 61,000) and the cuI(II1) mis of neonates and infants consisted of two CB8 (MW 12,000) were selected as marker peptides of apparently different molecular peptides of type I and III collagens, respec- weight. The first peak corresponded in mitively. The faster migration of the (~I(111) gration position to the cu1CB6 of bone and CB8 was not associated with an increase in the second peak, to the LYI CB6 of pepsin band width as was seen in homogenous solubilized type I collagen. Only the second 7.5% gels. Our finding of the ~y2CB3.5 in peak was obtained from adult dermis. The the 11% regions and the cuI(II1) CB8 in the carboxy-terminal (~1CB6 is known to con19% regions of the polyacrylamide concen- tain helical and nonhelical portions of which tration gradient suggested that these bands the latter is susceptible to cleavage by pepwere being continuously sharpened by this sin and acid conditions (8). The second (~1 gradient. Varying concentrations of type I CB6 peak probably contained the shorter and type III CNBr peptides over the range form (MW 18.000) and the first peak, the of 5 to 20 pg were applied in duplicate. Over more complete form of the peptide (MW this range a linear relationship existed be- 20.000). The reason for the age-related diftween the total concentration of type I or ference in the proportion of these two forms type III peptides applied and the densitome- of the LYI CB6 obtained from dermis was try areas of the a2 CB3.5 and the cuI(III) unclear. CB8. respectively. From eight runs we Insoluble dermis also yielded several exfound that the area of the 02 CB3.5 peptide tra peaks. One peak [peak ( I ), Fig. 21, which per microgram of total type I peptides probably contained two or more crossloaded was 68.2 _t 1.9 (SEM) and the area linked peptides (9), was not observed in our of the cuI(II1) CB8 per microgram of total soluble collagen or bone collagen preparatype III peptides loaded was 49.8 2 2.1 tion. In addition, a second peak [peak (2).

ANALYSIS

1.0. 7 K 1 :: e o-5

E ffl

m Q

COLLAGE~N

DEHMIS

~_\ ..

0 1.0.

./I

i tx BONE

1

MIGRATION

FIG 2. Electrophoretograms was identified from published cjf the

very

small

187

PEPTIDES

ia)

‘LL L‘ : m

OF

terminal

of CNBr data (8.9). pcptldes

peptides Peak (I)

(II-

(‘20 p*g) obtained probably contains

i

WI,

from (a) dermis a cross-linked

and peptide

(b)

bone. Each and peak (2).

peak some

(9.10).

Fig. 31 situated beyond the type Ill marker peptide was also observed in the bone but not in the soluble collagen preparations. It is likely that this peak contained the very small terminal peptides which are usually removed during pepsin solubilization of collagen f IO).

In contrast to dermis the samples of bone appeared to contain only type I collagen peptides (Fig. 3). Furthermore, the tr1 CB6 migrated with an apparently greater molecular weight than the tuI CB6 obtained from pepsin-solubilized type I collagen. The recovery of the tu1 CB6 was 86f4 of that obtained from soluble type I collagen indicating that in this bone preparation a llarge proportion of the cross-linkages involving this peptide were susceptible to acid cleavage. DISCUSSION The main object of improving the recoveries of the small peptides (MW 12.00010,000) was achieved using gradient gels. This improved recovery was probably the result of decreased diffusion of the peptides

in the more concentrated gel and the use of 50% methanol as the precipitant. No attempt was made to study the very small peptides although some of these were clearly evident in these gels. Comparison of published scans (6.10) of collagen CNBr peptides separated by electrophoresis on uniform 7.5 and IX cylinder gels confirm our finding that the recoveries of the small peptides are improved when a higher polyacrylamide gel concentration is used. However, the resolution of the slower migrating larger peptides appeared to be less when the 12V polyacrylamide gels were used. In this study we found that when gradient polyacrylamide gels were used improved recovery of the small peptides was combined with adequate resolution of the larger peptides. A further advantage of the gradient gels was the separation of the small type 111collagen peptides 3. 6. and 8. which usually migrate as one peak on 7.5 and I?? gels. into two peaks. Using gradient gels. bands containing less than I pg of type 1 or type III collagen peptides were readily detectable. As a consequence a qualitative assessmentof the presence of type I and type III collagen

188

COLE

AND

peptides could be readily made. A semiquantitative assessment of the relative amounts of type I and type III collagens could also be made by converting the densitometry areas of the a2 CB3.S (type 1 collagen marker) and the cxI(III) CB8 (type III collagen marker) into weight ratios using a calibration factor derived from the CNBr peptides of pepsin-solubilized collagens. Estimations of the relative proportions of type I and type III collagens and of type 1 and type II collagens using electrophoresis of CNBr peptides have been shown to correlate well with other methods of estimation ( I3,14). However, the method should only be considered to be semiquantitative because variations in the areas of the type I and type III marker peptides could result from differences in the completeness of the CNBr reaction. comigrating cross-linked peptides or consumption of the peptides in acid stable cross-linkages (9). This method of analyzing CNBr peptides has several features which suggest that it may be suitable for the study of small amounts of tissue collagen in human connective tissue disorders. These features include the ability to make a qualitative and semiquantitative estimation of type I and type III collagens and an assessment of some aspects of peptide involvement in cross-linkages.

BEAN

ACKNOWLEDGMENTS We arc indebted to Professor D. Danks and the Members of the Genetic’s Research Unit for their assistance. This work was supported by grants from the National Health and Medical Research Council of Australia. the Royal Children‘s Hospital. Melbourne. and +11,Australian Orthopaedic Association. LllC

REFERENCES I. Miller. E. J.. Epstein. E. H.. Jr.. and Piez, ( 1971 ) Bioc~ltetn. B;r,ph~.s. Rc,s. Comntrrrr.

K. A. 42,

1024-1029.

2. Epstein,

E. H..

Jr.,

( 1974) J. Bid.

Chum.

249,

3X-3231.

Muller. P. K.. Lemmen. C.. Gay, S., and Meigel, W. N. t 1975) Etcr. J. Bioc,hmr. 59, 97- 104. 4. Fujii. K.. Kajiwara. T., and Kurosu, H. (1977) FEBS 5.

Lcrr.

Furthmayr. c~hcm.

6.

Scott.

9. 10. Il. 12. 13. 14.

41,

P. G..

25 l-254.

and Timpl,

R. (1971)

Ano/.

Bio-

510-516.

A. G., and Veis. A. (1976) 25 l-257. Van Der Rest. M.. Cole. W. G., and Glorieux, F. H. (1977) BL,c~hrm. .I. 161, 527-534. Scott, P. G.. and Veis. A. t 1976) C‘onnt~f. ZJSU~ RCY. 4, 107-116. Scott, P. G.. and Veis, A. (1976) C‘~~nnrcr. 7ic.rur Rc,s. 4, I l7- 129. Weber. L.. Meigel, W. N.. and Rauterberg. J. (1977) Awk. Denrrcitol. RPS. 258. 25 l-257. Margolis. J.. and Kenrick. K. G. (1968)Anml. Bioc~hent. 25, 347-362. Chung. E.. Keele, E. M., and Miller. E. J. (1974) Bbdzr~rnisfr~ 13, 3459-3464. Eyre. D. R., and Muir, H. t 1975) C~vtnrct. Ti.s.trrt, Rr.r. 4, I I - 16. Eyre. D. R.. and Muir. H. (1976) Eiwhem. ./. 157, AM/.

8.

82,

H..

267-270.

Telser.

Bioc~hrm.

70,