Studies on the Chemistry and Fine Structure of Elastic Fibers from Normal Adult Skin* David P
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Till:: JOURNAL Ot" I NVf~ST I GAT I VE DEilMATOLOCY Vo l. 59, No. 3 Copy ri ght © 1972 by The \Vill inms & \Vilkins Cu. Printed in U.S ..4.. STUDIES ON THE CHEMISTRY AND FINE STRUCTURE OF ELASTIC FIBERS FROM NORMAL ADULT SKIN* DAVID P. VARADI, M.D., F.R. C. P. (C) ABS TRACT Collagenase-prepared, purified elastic fibers from adult huma n skin consist of at least two morphologically distinct components, the unstained a morphous component, and inter nally-located, deeply staining microfibrils. Separation a nd isolation of the amorphous component was achieved by alka li preparation of the elastic fibers. E lectron micrographs of a lkali prepared fibers showed essentia lly a morphous componen t as most of the microfibrils had been solubilized. The a mino acid composition of the amorphous component was that of classical elastin. The microfibrillar component embedded deep wi thin the interstices of the fiber was sepa rated by, first, re movin g the a morphous component with elastase, a nd subse quently solubilizing, with dithioerythritol (DTE), the cystine-containing microfibrils con tained in the elastase-produced residue. The amino acid composition of this DTE-ex t racted microfibrilla r material was similar to that of the peripheral microfibrillar compo nent enzymatically removed from bovine fetal elastic fibers. Electron microscopi c moni toring showed that only stained microfibrils remained after elastase digestion. A protein portion of the microfibrilla r component was not solubilized by the DTE and was presumed to contain a low concentrat ion of disulfide bonds. Studies on purified elastin (amorphous component) revealed that a lanine is concentrated around desmosine crosslinks and that the pyrollidines a re uniformly distributed a long t he elastin molecule thereby precluding a a-helix conformation. Dark fi eld electron mi croscopy suggested that the desmos ine cross link region is dumbell -s haped and t hat t he desmosine crosslinks a re not equidistant from each other on the peptide chains. It has been established by electron microscopy cated microfibrils in large numbers. This investi that mature elastic fibers, after staining with cati gation reports on a) the separation of microfibril onic lead and uranyl acetate, consist of a centra l from a morphous component, b) the chemical na non-staining amorphous core surrounded by ture of adult microfibrils in relation to those iso stained tubular appearing microfibrils (1, 2) . In a lated from feta l elastic fibers, c) the distribution recent study on collagenase-prepared elastic fi of some of t he amino acids in alkali -prepared bers from fetal bovine li gamentum nuchae, the elastin (amorphous co mponent) and d) the fine central amorphous component was separated structure of acid-solubilized elastin examined by from the periphera l microfibrillar component by da rk fi eld electron microscopy. solubilizing t he cystine-containing microfibrils with the reducing compound, dithioerythri tol (3). MATERIALS AND METHODS Partial characterization of the separated compo nents showed that the amorphous component was Isolation and purification of elastic fib ers. A non -hy t he desmosine-containing protein, elastin, while drolyti c collage nase meth od of Miller et al. (4) was used the microfibrils consisted of protein(s) with a n to isolate elastic fib ers from norm al adul t human skin. Necropsy specimens of skin we re obtained from the a mino acid composition quite different from that upper, outer thi gh of male ca davers aged forty to sev of elastin. enty-two years . The spec imens obta ined from three di f In the present study on elastic fibers from ferent cadavers were processed se parately and were not norma l adult human skin, it was noted that elec pooled at any stage . Fat and epiderm is were mechani tron micrographs of such elastic fibers showed ca lly removed from the skin. The remaining dermis, cut essentially no peripheral but only internally-lo- into small pieces, was fin ely min ced in 3% Na 2 HPO, with a Sorva ll Omni-Mixer. Treatment with 25% KCI , 5 Received Jan uary 7, 1972; accepted for publication M ga unidine HCI and collagenase was ca rri ed out ac May 2, 1972. co rding to the method of Miller et a.l. (4). This work was supported by the Medi cal Research A portion of the above prepared elastic fib ers was Coun cil of Canada (MA-3185) and the Womens Auxi l fur ther treated with an alkali method in whi ch the fi iary of the Welles ley Hospital. bers were suspended in 0.1 N NaO H at 98 ° C for one Part of thi s wo rk was prese nted at the annual hour. Prior to this treatment the fibers had bee n left in meeting of The American Federation for Clini cal Re acetone and then ether, 24 o C, for 24 hours, respec search in Atlantic City on May 3, 1970. An Abstract was tively. pub lished in Clini ca l Research, 18: 352, 1970. * From the Department of Medicine, Section of Der Electron mi crosco pi c moni torin g of the collage nase matology, Wellesley Hospital, University of Toronto, prepared and alkali -treated elastic fibers showed the Toronto, Ontario, Ca nada. former to contain both mi crofibrils and amorphous 238 CHEMISTRY AND FINE STRUCTURE OF ELASTIC FIBERS 239 component while t he latter consisted of essentially stained, unshadowed macromolecules of biological or amorpous co mponent devoid of micro!ibrils. igin . For examination, the samples of solubilized elastin Elastase diges tion. Elastin preparations were in cu were dissolved in cold 0.01 M phosphate buffer, pH 7.0, bated with electrophoretica lly purified elasta e (Wor at conce ntration of 1 ng/ m I. thington Biochemica l Co rp. ) in 0. 1 M glyci ne buffer (pH Chemical analyses. Am in o acid analyses carried out 7.0) at 37° C for 48 hours. One mg of elastase was used by the Beckman-Spinco Auto analyser were performed fo r every 5 mg of elastin (4). on samples of elastin hydrolyzed in 6 N HCI in vacuo Solubilization of microfibrils fr om adult dermal for seventy-two hours at 106° C. Desmosine and isodes elastic fibers. The res idue remaining after elastase mosine, respectively, were quantitatively determined on digestion of co ll agenase-prepared dermal elastic fibers the analyser according to a procedure by Anwar (8). was treated with the reducing agent, di thi oerythritol Microfibrillar m aterial was hydrolyzed 24 hours. (DTE), to so lubilize the microfibrils by reduction of di Protein concentrations of the oxali c acid solubilized sulfide bonds. The reduction procedure is that of R oss elastin fractions were determined by the method of and Bornstein (3). On dialysis, so me of the solubilized Lowry using solubilized elastin from bovine li gamentum material prec ipi tated out. The still solubilized portion nuchae as the standard. of microfibrillar co mponent was des ignated the 'super natant'. RESULTS Preparation of soluble e lastin fra ctions. Soluble elastin was prod uced by partial acid hydrolys is of alkali Chemical Studies prepared elastin in boiling 0.25 M oxali c acid solution T he amino acid com posit ion of hot alkali ex according to the method of Partridge, Dav id and Adair tracted elastin from the t high skin of a seventy (5). After treatin g t he elastin with the oxali c acid solu year-old ma n i s s hown in Table I. T h tion in a boiling water bath for a half-hour, t he so lution e composi was removed and water washings of the remaining inso l t ion is characteristic for elastin. An a lmost iden uble elastin added to it. This was des ignated E xtract 1. tical composition was obtained o n elastin isolated Fresh oxali c acid solu tion was added to the res idual in from t he s kin of a forty-three-year-old and s ixty soluble elastin and boiling was carried out for one hour. year-old subject, respectively. In the same table Extract 2 was then removed. Subsequently, every hour a re g iven the a mino acid compositions of the 6 for fou r m ore hours the extracts with their washings fractions extracted with oxalic acid solut ion from were removed and fresh solution added. After 5 1!2 hours insolubile elastin. There is a steady increase in the insoluble elastin had dissolved to fo rm 6 extracts the isodesmosine a nd desmosine concent rations numbered 1 to 6. Extracts 4 and 5 which had a hi gh a from Extract 1 to 6 while certain amino acids elastin co ntent, were each chromatographed at go C on such as a Sephadex G-100 column equilibrated with 0.05 M proline, valine, leucine, phenylalanine phosphate buffer containing 8 M urea. The materi al and arginine remain relatively constant. Alanine emergin g in the vo id volume, the a-elastin, was selected shows a stead y increase unt il in fraction 6, the for d a rk fi eld electron microscopy. alanine exceeds glycine in concen tration. The The elastin fragments shown in Fi gures 7, 8, 9 were a aspartic acid a nd glycine tended to decrease in gift from Dr. R. A. Anwar. This material, designated amount. The increase in concentration of the Dowex V in a recent publication by Shimada, Bowman, des mosines probably indicates progressive hydro Davis and Anwar (6), was obtained by applyin g a n elas lytic cleavage of peptide chains with proportion tase digest of bov in e li ga mentum nuchae elastin se a ll y less extraction of t he desmosine linkage re quentially to Sephadex G-25, cellulose phosphate and gions t hereby Dowex 50W co lu mns.