Coacervation and ion-binding studies on aortic elastin

Biochimica et Biophysica Acta, 31o (1973) 481-486 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 36424 COACERVATION AND I O N - B I N D I N G S T U D I E S ON AORTIC E L A S T I N

BARRY C. STARCHER, GAETANO SACCOMANI AND DAN W. U R R Y

Laboratory of Molecular Biophysics, University of Alabama Medical Center, Birmingham, Ala. 35294

(U.S.A.) (Received November 7th, 1972) (Revised manuscript received March 6th, 1973)

SUMMARY

CD studies were carried out on porcine aortic elastin in solution and as the coacervate. As with elastin from bovine ligamentum nuchae, coacervation results in a marked conformational change; addition of sodium dodecyl sulfate to an aqueous solution of aortic elastin mimics the effect of coacervation; dissolution in trifluoroethanol most closely reproduces the coacervate CD pattern, and addition of CaC12 to the trifiuoroethanol solution causes a conformational change demonstrating ion binding. The inverse temperature transition which is responsible for coacervate formation at body temperatures and the effect of sodium dodecyl sulfate and trittuoroethanol in producing the coacervate CD pattern in solution suggests that hydrophobic interactions are important in the ordered association of a-elastin molecules to form the coacervate.

INTRODUCTION

Calcification of the aorta localizes primarily on the elastic fibers1, ~. Although calcification of aortas occurs frequently and can be induced in experimental animals3, 4, the process is poorly understood. As has been pointed out previously 5, implication of functional side chains as major sites in initiating calcification is unlikely due to the extremely low concentrations of these groups in elastin compared to the relative affinity of this protein for Ca ~+. Recent studies, using CD and NMR to study conformational changes in elastin and other model compounds, have led to the proposal that elastin binds Ca 2+ at neutral sites, utilizing the acyl oxygens along the peptide backbone to coordinate the Ca 2+ (ref. 5). These studies, as well as others concerned with conformational changes, have been conducted on elastin purified from bovine ligamentum nuchae. While chemically very similar, ligamentum nuchae could be quite different functionally and structurally from aortic elastin. The present study was conducted to determine if porcine aortic elastin undergoes the same conformational changes seen with ligamentum nuchae elastin, and to further understand the processess of ion binding and coacervation.

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METHODS

Elastin was purified using essentially the method of Ross and Bornstein 6. 3-month-old pig aortas were obtained locally (courtesy of Lumberjack Meats, Inc.), cleaned of adhering tissue and adventitia, and homogenized in a Waring blender in 3 vol. of o.9% NaC1. The homogenized aorta was stirred overnight in the cold, centrifuged, and the supernatant discarded. The residue was resuspended in o.9% NaC1 and extracted over another 24-h period. After centrifugation, the residue was suspended in 3 vol. of 5 M guanidine. HC1, pH 7.5, homogenized, and extracted for 24 h in the cold. This process was repeated two more times on the residue after centrifugation. The final residue was washed with water, suspended in 3 vol. of water, and autoclaved for 2o min. This process was repeated, usually 5 times, until there was no soluble protein in the supernatant. The residue, which at this point was almost pure elastin, was incubated at 37 °C for 24 tl in I vol. of 2% (NH4)2COa containing I mg trypsin per g of original aorta. At the end of the incubation period, the elastin residue was washed with water and freeze dried. Soluble elastin was prepared by the method of Partridge et ald, using hot oxalic acid extractions, which produces two protein fractions; a-elastin (tool. wt 7o ooo) and fl-elastin (tool. wt < io ooo). Rather than dialyze the protein to eliminate the oxalic acid and risk losing the fl-elastin, the combined oxalic acid extracts were freeze dried, dissolved in a small volume of water, and the protein separated from the oxalic acid on a 2.5 cm × IOO cm column of Sephadex G-25. The protein fraction was freeze dried, redissolved in a small volumn of water and separation of the aelastin from the fl-elastin was achieved on a 2.5 cm × IOO cm column containing Sephadex G-75, equilibrated with o.oi M acetic acid. Fractions containing the proteins were pooled and freeze-dried. Amino acid analysis was performed on a Beckman Model 116 analyzer by the procedure of Starcher 8. Elastin was hydrolyzed in 6 M HC1 for 72 h in sealed evacuated tubes. No correction was made for amino acid destruction during hydrolysis. Coacervation of aortic elastin was achieved as described previously 9. Protein concentration was calculated from amino acid analysis of the coacervate film after elution from the cell surface. The CD spectra were obtained on a Cary Model 6o Spectropolarimeter equipped with a Model 6OOl CD and simultaneous absorption accessories. Protein concentration was calculated from amino acid analysis. RESULTS AND DISCUSSION

Having in mind that one would like to rebuild the findings and concepts to apply them to the in vivo situation, one of the difficulties in purifying elastin for use in physical studies is treating the protein as gently as possible and yet achieving a high degree of purity. Extraction of elastin with hot NaOH achieves purity, but the extent of hydrolysis and possible rearrangement is unknown. For this reason we chose to purify the elastin without NaOH treatment, using as our criterion of purity the amino acid analysis, with special emphasis on the absence of histidine and hydroxylysine. The amino acid analysis of the purified elastin, fl-elastin, a-elastin and its coacervate is shown in Table I. The only major difference in amino acids between

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COACERVATION AND ION BINDING OF ELASTIN TABLE I

AMINO ACID ANALYSIS OF PORCINE AORTIC ELASTIN, (I-ELASTIN, ~-ELASTIN AND THE COACERVATE F R O M ~-ELASTIN V a l u e s for a m i n o acids are e x p r e s s e d as r e s i d u e s p e r i o o o residues.

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4.8 12.8 9-3 I9.4 119 329 231 o 13o o 18.7 48.1 i6.4 30.6 7.2 8.9 6.4 o 6.3

4-3 15.4 i I. I 21.7 117 305 261 o 116 o 17. i 53.8 2o.4 29-4 8. I lO.7 5.3 o 5.2

4 .6 12.7 9.2 17.6 112 341 231 o 133 o 17.9 52.6 14.8 3 o.1 6.3 7.3 5.7 o 5-2

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the groups was a higher content of alanine and less glycine and valine in the a-protein and its coacervate. Another difference lies in the degree of crosslinking demonstrated by the ratio of the desmosines to lysine, this ratio being much higher in the a-protein and its coacervate than in the whole protein or the E-fraction. These findings are consistent with the recent sequencing data by Sandberg et al. 1°, who have found various repeat sequences occurring in elastin, with the area of potential crosslinking being rich in alanine. A mean residue weight value of 82 g/mole, used in ellipticity calculations, was taken from the amino acid analysis of the coacervate. Fig. I shows the CD curves of a-elastin in solution and of the coacervate and the corrected spectrum for the coacervate n. As was demonstrated with ligamentum nuchae, molecularly dispersed aortic elastin exhibits a negative band near zoo nm commonly considered typical of disordered proteins. On coacervation aortic elastin undergoes a conformational change demonstrated by the red shift of the large negative band to 2o 7 nm, by enhancement of the negative band near 222 nm and by the formation of a large positive band near 192 nm. The resultant CD pattern is typical of proteins and polypeptides containing about 2o-25% a-helix. The results could be due to formation of a-helix in the alanine-rich regions of crosslinking, to an unmasking of a-helix already present, or in analogy to gramicidin S to the formation of fl-turnsa~, 13. It is now apparent that elastin in the native state possesses a certain degree

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of order. Early studies by Gotte et a l ) 4 and more recent high resolution electron microscopy studies (Gotte, L. and by Quintarelli, G., personal communication), using a different method of preparation and staining, have raised the question that elastin is not the amorphous substance that has been described for years 15, but rather is composed of well defined fibers. The unusual property of coacervation that elastin possesses gives an indication of a force responsible for the order that is present. Coacervation likely results from hydrophobic interactions between two adjacent polypeptide chains, since increased temperature, which in aqueous systems enhances hydrophobic interaction, promotes coacervation. We have found that addition of dimethylsulfoxide, which disrupts hydrophobic interactions without disrupting hydrogen bonding, will prevent the formation of the coacervate. This corresponds well with X-ray diffraction studies of elastin 14 showing a series of diffuse rings. One of these rings at 8. 9 A disappears on addition of dimethylsulfoxide. In a forthcoming paper (Urry, D. W., to be published) a conformational model will be proposed for a repeat sequence of elastin that explains the loss of the 8. 9 A repeat as the separation of fl-spirals 18 held together largely by hydrophobic forces. Since the coacervate forms at body temperature and possesses a definite order, we have proceeded on the assumption that this conformation is relevant to elastin in vivo. Thus the coacervate provides a model CD pattern which we would like to obtain in a soluble state in order to study further elastin interactions. Fig. 2 shows the effect of 0.05 M sodium dodecyl sulfate on a-elastin in water. There is observed a change in the spectra similar to that observed on coacervation. Ligamentum nuchae elastin has been reported to give the same CD shift in tile presence of sodium dodecyl sulfate (Kagan et al.17). Problems occur when studying

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Fig. 2. CD patterns of a-elastin. Curve a is in o.oi M sodium acetate, Curve b is in the presence of o.o 5 M sodium dodecyl sulfate. Fig. 3. CD patterns of a-elastin in trifluoroethanol containing 2.5% water (Curve a). Curve b shows the effect of addition of CaC12 (1.5 8 mg), Curve c the effect of addition of MgC12"6H20 (2.89 mg). i n t e r a c t i o n s of elastin in t h e presence of sodium d o d e c y l sulfate, e.g. ion binding, since the s o d i u m d o d e c y l sulfate, b o u n d to the protein, carries a s t r o n g n e g a t i v e charge c a p a b l e of i n t e r a c t i o n with cations. R e c e n t l y it has been shown t h a t l i g a m e n t u m nuchae elastin can also achieve this o r d e r e d c o n f o r m a t i o n in trifluoroethanol is. Trifluoroethanol often solubilizes m e m b r a n e p r o t e i n s in an average o r d e r e d s t a t e n o t g r e a t l y different from the s t a t e within t h e m e m b r a n e 19,2°. Also since the c o a c e r v a t e is f o r m e d on raising the t e m p e r a t u r e , this inverse t e m p e r a t u r e t r a n s i t i o n in an aqueous m e d i u m is i n d i c a t i v e of h y d r o phobic i n t e r a c t i o n s in t h e coacervate, i.e. a s i t u a t i o n analogous to h y d r o p h o b i c intera c t i o n s of p r o t e i n s w i t h i n m e m b r a n e s . These two p o i n t s t a k e n with the CD d a t a suggest trifluoroethanol as a r e l e v a n t solvent for s t u d y i n g i n t e r a c t i o n s in a solubilized state. L i g a m e n t u m n u c h a e elastin in trifluoroethanol r e a d i l y i n t e r a c t s with Ca 2+, p r e s u m a b l y at t h e acyl o x y g e n s i8. T h a t this is also true of aortic elastin is shown in Fig. 3. Ca2+ induces a reversal to the aqueous t y p e CD s p e c t r u m . Mg 2+ causes a similar, b u t m u c h smaller shift. Sr 2+, B a ~+, a n d Be ~+ were also s t u d i e d for their a b i l i t y to i n t e r a c t with elastin in trifluoroethanol. Be 2+ h a d no a p p a r e n t effect, while SI ~+ was a l m o s t as effective as Ca 2+. B a 2+ h a d some effect at low concentrations, b u t was insoluble a t the higher c o n c e n t r a t i o n s used w i t h Ca 2+ a n d Sr 2+. The fact t h a t Mg 2+ i n t e r a c t s with elastin is interesting in light of the findings t h a t m a g n e s i u m deficiency results in a m a r k e d increase in calcification of the aorta, the calcification being localized on the elastin fiber a,21. The a m o u n t of Ca 2+ t h a t i n t e r a c t s with elastin in a pure aqueous s t a t e is v e r y small, in t h e order of I Ca 2+ per 5oo residues a n d is p H d e p e n d e n t 22. This would suggest b i n d i n g at c h a r g e d sites, however, once Ca 2+ b i n d i n g d i d occur at these sites the charge would be n e u t r a l i z e d a n d would leave no d r i v i n g force to a t t r a c t PO48a n d s t a r t m i n e r a l i z a t i o n . If, on t h e o t h e r h a n d , elastin were in t h e ordered conform a t i o n as seen in t h e coacervate, a c o n f o r m a t i o n which has been shown to i n t e r a c t

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with Ca 2+ in organic solvents, then Ca 2+ binding to neutral sites would be an excellent initiator of hydroxyappatite formation. The elastin matrix would become positively charged with Ca ~÷ which would then attract phosphate counterions. The driving force would be the affinity for neutral sites, the propagating force would be the attraction of counterions allowing further charging of the elastin matrix. ACKNOWLEDGMENTS

This work was supported by National Institutes of Health, Grants HE-I45IO and H L - I I 3 I O , and by the Mental Health Board of Alabama. We wish to thank Miss L. Caudill for technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 1o i1 12 13 14 15 i6 17 18 19 20 21 22

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