Chemical characterization of α-elastin components

Biochirnica et Biophysica Acta, 365 (1974) 115-120

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36790 CHEMICAL CHARACTERIZATION OF tt-ELASTIN COMPONENTS GIOVANNI ABATANGELO, ROBERTA CORTIVO, DANIELA DAGA-GORDINI and GUIDO GARBIN Istituto di lstologia ed Embriologia Genera& dell' Universitd di Padova, Padova (Italy)

(Received February 27th, 1974)

SUMMARY Using an SE-Sephadex column, it was possible to separate five peptide fractions from a-elastin. Each fraction has been separately analyzed in order to determine the amino acid composition, the N-terminal residues and the homogeneity on Agarose gel and by disc electrophoresis. The amino acid composition of the single fractions reveals no significant differences when compared with native elastin. Serine, glutamic acid, glycine and alanine are the only N-terminal residues present. The absence of other Nterminal residues has been interpreted as due to a selective cleaving by oxalic acid which probably occurs only at specific amino acids. As far as the homogeneity of the single fractions is concerned, the data obtained with agarose gel and by disc electrophoresis, indicate that these fractions are not pure but mixtures of microdispersed peptides.

INTRODUCTION Elastin is a highly insoluble connective tissue protein and, therefore, all chemical and physicochemical studies are necessarily confined to soluble fragments obtained by various degradation procedures. The solubilization of elastin can be achieved by a number of methods, e.g. alcoholic KOH hydrolysis [1], controlled elastolysis, treatment with strong mineral acids or by hydrolysis with hot 0.25 M oxalic acid as described by Partridge et al. [2]. A comparative study of the hydrolysis of bovine ligamentum nuchae elastin by various specific enzymes and by alcoholic KOH has been reported by Mandl and co-workers [3, 4]. These methods have been found to yield soluble products in all cases, but with important differences in molecular weight distributions and amino acid compositions. It has been shown [5] that tt-elastin obtained by 0.25 M oxalic acid hydrolysis [2], is composed of several fractions. Podrazky [5] using an SE-Sephadex column, has been able to separate 7 fractions from the elastin coacervate of the elastin digest, thus demonstrating the heterogeneity of this soluble material. This preliminary observation on the heterogeneity of the tt-elastin, however, did not give any information

116 about the amino acid composition, molecular weight distribution and homogeneity of the single fractions. Moreover, given the extreme importance of the soluble products of elastin, which allow the study of this protein from chemical and physicochemical points of view, it is very useful to characterize these components in order to make comparisons with the parent protein. We, therefore, thought that it was worthwhile to try some chromatographic procedures which would allow the characterization of the c~-elastin components. In this paper the chromatographic separation of solubilized ~-elastin is reported. The chemical characterization of the single fractions obtained and their molecular weight distributions are also studied. MATERIALS AND METHODS

Preparation of purified elastin The autoclave-purified elastin was obtained from ligamentum nuchae of 1-2year-old calves according to the procedure described by Partridge [2]. Soluble elastin was obtained by 0.25 M oxalic acid hydrolysis as described by Partridge. The hydrolysate was dialyzed against distilled water and then adjusted to pH 4.7, 0.01 ionic strength, to obtain coacervation [2]. The coacervate was collected and was lyophilized after dialysis. This material (e-elastin) represented 40),~, of the original elastin.

Chromatographic separation of the a-elastin components The a-elastin obtained as above was fractionated on an SE-Sephadex column as follows: Aliquots of SE-Sephadex C-25 were suspended in 0.2 M acetic acid-0.2 M sodium acetate buffer at pH 3.65 and then packed in a 4 cm × 100 cm glass column. 300-500 mg of a-elastin was applied on the column and then eluted with 0.2 M acetic acid-0.2 M sodium acetate buffer (pH 2.6-6.0) with a linear gradient, followed by 0.1 M NaOH. The single fractions were separately collected and lyophilized.

Desalting of the peptide fractions The single fractions obtained from the SE-Sephadex column were dissolved in water and then applied to a 3 cm × 80 cm column of Bio Gel P2 200-400 mesh and eluted with water in order to retard the salts.

N-terminal residue determination The determination of the N-terminal residue was performed by dansyl [6, 7] and dinitropyridyl [7] methods.

Molecular weight determination For the molecular weight determination two different methods were adopted: column chromatography with Bio Gel A-1.5 (200-400 mesh) and disc electrophoresis according to the method described by Davis [9]. The Bio Gel A-1.5 (200-400 mesh) was equilibrated with 0.1 M NazHPO4-NaH2PO4 buffer (pH 7.4) and then packed in a 2 cm × 150 cm glass column. As internal standards, aldolase, bovine serum albumin, egg albumin, chymotrypsinogen A and myoglobin were used.

117 fl-Mercaptoethanol was added beforehand to the samples to a final concentration of 1 ~ .

Amino acid analysis For the amino acid analysis, the samples were hydrolyzed with 6 M HCI at 110 °C for 22 h and then applied on the amino acid analyser JLC-5AH. RESULTS In Fig. 1 the separation of the a-elastin on the SE-Sephadex C-25 column is shown. Five main fractions were obtained, the first three of these were eluted with the II o

2

V~

IV ~ -~

ii1~

t.___/

.~ o.a cu• 0.4 I - - ~ ~ o ~ 0.2

~1 ~

1

12

~ 14

l'e

II

~ 30

32

r 34 ml -~0~

~

Acetate

Buffe~

-

-

N a O H - -

Fig. 1. Chromatographic separation of the ct-elastin components on the SE-Sephadex C-25 column (4 cm × 100 cm). The arrows indicate the portions of the Fraction III which were separately collected in order to determine the amino acid composition (see Table II). acetate buffers and the last two were eluted by 0.1 M N a O H . Each fraction was separately collected and then desalted on a Bio Gel P2 column (see Materials and Methods). In Table I the amino acid analyses of all five peaks are reported. The data indicate that there are pronounced differences in composition between the various fractions as far as the amount of the polar amino acids is concerned. This difference has been expressed by the ratio of the nonpolar to polar amino acids. Because Fraction SE I I I was eluted as a broad peak, three different tubes were collected during the chromatographic elution (as indicated with the arrows in Fig. l) in order to determine the amino acid composition. In Table lI the amino acid analyses of the three single tubes is reported. No significant differences in amino acid composition were observed and the Peak SE | I I was collected as a single fraction.

118 TABLE 1 A M I N O ACID COMPOSITION OF P U R I F I E D ELASTIN AND PEPTIDES OBTAINED BY ~t-ELASTIN F R A C T I O N A T I O N Amino acid residues per 1000 residues separated on the SE-Sephadex column. Amino acid

SE 1

SE I1

SE 111

SE IV

SE V

Elastin

Hyp Asp Thr Ser Glu Pro Gly Ala Val lie Leu Tyr Phe lsodesmosine Desmosine Lys His Arg NP/P*

12.3 4.7 6.7 7.1 14.4 108.5 352.9 233.2 140.0 21.2 58.6 6.8 27.0 1.0 1.2 2.1 • 3.4 23.8

12.4 5.2 8.1 9.3 15.2 112.0 341.0 248.0 121.0 19.8 58.0 n.c. 30.7 2.4 3.0 2.9

10.0 4.2 8.5 10.6 17.9 106.5 317.5 250.8 150.9 21.2 57.0 5.0 29.7 3.2 5.5 3.2

4.6 19.8

5.1 18.2

10.4 7.4 10.1 11.2 18.3 104.4 307.0 239.6 149.0 20.7 58.6 13.9 30.1 2.5 3.0 4.9 1.7 7.2 14.4

10.1 11.8 12.7 14.0 25.8 109.4 306.5 248.3 113.5 23.6 62.1 10.2 32.7 1.9 2.6 4.9 2.0 8.2 10.8

7.1 6.0 6.4 6.4 15.9 117.2 328.3 242.0 133.7 25.2 62.8 7.8 30.8 1.7 2.3 4.0 0.5 6.0

~ NP/P, ratio of nonpolar to polar amino acids.

In T a b l e I11 the N - t e r m i n a l residues o f e a c h f r a c t i o n e l u t e d f r o m the SES e p h a d e x c o l u m n are r e p o r t e d . Serine, g l u t a m i c acid, glycine a n d a l a n i n e were f o u n d to be t h e N - t e r m i n a l residues p r e s e n t in all f r a c t i o n s . T h e p r e s e n c e o f the s a m e a m i n o acids as N - t e r m i n a l in all t h e f r a c t i o n s m a y suggest a selective c l e a v i n g m e c h a n i s m by o x a l i c acid. In Fig. 2 the e l u t i o n profile o f v a r i o u s s t a n d a r d p r o t e i n s on the B i o - G e l A-1.5 ( 2 0 0 - 4 0 0 m e s h ) c o l u m n is r e p o r t e d . O n this c o l u m n t h e single f r a c t i o n s s e p a r a t e d o n t h e S E - S e p h a d e x c o l u m n were a p p l i e d (Fig. 1) in o r d e r to s t u d y the m o l e c u l a r w e i g h t d i s t r i b u t i o n s . A single l a r g e p e a k was o b t a i n e d for e a c h f r a c t i o n c o r r e s p o n d i n g to a wide r a n g e o f m o l e c u l a r weights. F r a c t i o n s S E I a n d I1 s h o w e d a large p e a k w i t h

TABLE 11 N - T E R M I N A L RESIDUES Residues present in the five fractions eluted from the SE-Sephadex column, expressed as g.moles/10z g of peptide. Amino acid Ser Glu Gly Ala

SE I

SE I1

SE llI

SE IV

SE V

70.0

30.2

27.1

103.4

112.0 457.2

62.4 123.1

218.3 24.1

83.0 42.1

58.0 54.1 56.5 132.2

119 TABLE III AMINO ACID COMPOSITION OF THREE DIFFERENT TUBES COLLECTED FROM PEAK SE III See Fig. 1. Amino acid

Tube I

Tube II

Tube III

Hyp Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe Lys Arg Isodesmosine Desmosine

14.5 5.5 8.9 12.9 17.8 137.9 295.2 245.2 124.7 23.2 60.4 5.3 31.0 4.5 7.7 2.3 2.2

10.8 4.5 8.3 11.2 17.3 134.0 299.8 248.8 130.4 23.5 54.5 5.2 29.6 5.4 6.5 2.0 2.9

12.0 4.6 8.4 10.2 17.8 133.1 286.7 258.6 141.7 23.9 60.8 4.7 30.6 6.1 6.3 2.5 3.4

a molecular weight range from 33 000 to 70 000. Peaks SE III, IV a n d V gave a larger peak with a molecular weight range from 40 000 to 160 000. By disc electrophoresis, behaviour c o m p a r a b l e to that obtained with the Agarose gel c o l u m n was seen as far as the molecular weight distribution is concerned. In fact on polyacrylamide gel the single fractions gave a large single b a n d related to a molecular weight, of the same value as that calculated with the Agarose column.

1.o

.a

E ;

._g

S~ ~

~ ~

.,

'~-~

,~

~

-~

"~

~ ~8 ~ ok

J\ ~

2~0

380

480

i

i

sso

i

~ 6so

i ?~o

i

81.o

i

Fig. 2. Chromatographic separation of some standard proteins on the Sephadex A-15 column (2 cm × 150 cm). The single fractions separated on the SE-Sephadex column were separately applied onto the same column in order to determine the molecular weight.

120 When the single fractions were applied to different c h r o m a t o g r a p h i c columns, in order to improve the analytical resolution, no positive results were obtained. Sephadex G-75 and G-100, QAE-, D E A E - and CM-Sephadex were used with unsuccessful results. DISCUSSION The only successful method for the fractionation of the ~-elastin was SESephadex where five main fractions were obtained. The amino acid analyses indicate a varying composition as far as the a m o u n t o f the polar amino acids is concerned. The sum o f the moles of proline, glycine, alanine, valine, isoleucine and leucine was divided by the sum of the moles of lysine, histidine, arginine, aspartic acid, threonine, serine and glutamic acid. This ratio, as can be observed, decreases from Peak SE I to SE V due to the progressive increase of the polar residues. It can also be observed that both basic and acidic amino acids increase proportionally. The three neutral a m i n o acids glycine, alanine and proline are present in an approximate ratio of 3/2/I in all five fractions, closely resembling the native elastin. O f particular interest is the presence o f specific N-terminal residues found in all fractions. Namely, only serine, glutamic acid, glycine and alanine were found to be present. O f these, glutamic acid was present only in the last fraction. The absence of other N-terminal residues such as proline, valine and leucine, which represent approximately one third of the amino acid composition, could suggest a selective cleaving mechanism o f the oxalic acid [10]. F r o m these data it can be concluded that the ct-elastin represents a very heterogeneous material, which, as already demonstrated by other investigators [5], can be roughly separated on a SE-Sephadex column. In addition we have demonstrated that these single peaks have to be considered as a microdispersed variety of peptides. F r o m this point of view, the use of these soluble materials, as an alternative to the insoluble native elastin, represents a limitation for chemical and physicochemical studies, even t h o u g h from the amino acid composition, these fractions are comparable with the parent protein. ACKNOWLEDG M ENTS We are most grateful to Mr Giorgio Michelotto for his technical assistance. The financial support of the Italian C N R is gratefully acknowledged. REFERENCES 1 Robert, L. and Poullain, N. (1963) Bull. Soc. Chim. Biol. 45, 1317-1324 2 Partridge, S. M., Davis, H. F. and Adair, G. S. (1955) Biochem. J. 61, 11-20 3 Mandl, I., Keller, S. and Levi, M. (1970) in Chemistry and Molecular Biology of the lntracellular Matrix (Balazs, E. A., ed.), Vol. 1, pp. 657-664, Academic Press, London 4 Keller, S. and Mandl, I. (1973) Connect. Tissue Res. 2, 49-56 5 Podrazky, V. (1968) Biochim. Biophys. Acta 160, 277-279 6 Gray, W. R. and Hartley, B. S. (1963) Biochem. J. 89, 379-380 7 Gray, W. R. and Hartley, B. S. (1963) Biochem. J. 89, 59P 8 Signor, A., Previero, A. and Terbojewich, M. (1965) Nature 205, 596-597 9 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404427 10 Gray, W. R., Sandberg, L. B. and Foster, J. A. (1973) Nature 246, 461-466