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Comparative biochemistry of collagen some amino acids and carbohydrates
METABOHSM OF Sarcina lutes. III
VOL. 30 (1958)
293
* J. B. LEATHES AND H. S. RAPER, The Fats, L o n g m a n s , G r e e n & Co., L o n d o n , 1925, p. 58. x0 W. R. BLOOR, J. Biol. Chem., 17o (1947) 671. 11 R. B. ROBERTS, D. B. COWIE, P. H. ABELSON, E. T. BOLTON AND R. J. BRITTEN, Studies o[ Biosynthesis in Escherichia cull, Carnegie I n s t i t u t i o n of W a s h i n g t o n , 1955. 12 S. DAGLEY AND J. SYKES, p e r s o n a l c o m m u n i c a t i o n . 13 S. DAGLEY, E. A. DAWES AND G. A. MORRISON, J. Sen. Microbiol., 4 (195 °) 437. i4 D. E. HUGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 15 L. H. STICKLAND,Biochem. J., 64 (1956) 498. is E. A. DAWES AND W. H. HOLMS, Biochim. Biophys. Acts, 29 (1958) 82. 17 W . SAKAMI,Handbook o] Isotope Tracer Methods, W e s t e r n R e s e r v e U n i v e r s i t y , Cleveland, Ohio. x955. is p. H. KOPPER, J. Bacteriol., 63 (1952) 639. x9 p. H. KOPPER, J. Bacteriol., 67 (1954) 5o7 • 20 D. JERCHEL AND H. FISCHEr, Ann., 563 (1949) 2oo. 21 F. C. NORRIS, J. j . R. CAMPBELL AND P. W. NEY, Can. J. Research, C27 ([949) 15721I j . LASCELLES, Biochem. J . , 62 (1956) 78. ~8 R. COOPER, Biockem. J., 63 (1956) 2 5 P . H. K. SCHACHMAN, A. B. PARDEE AND R. Y. STANIER, Arch. Biockem. Biophys., 38 (1952) 245. H. HALVORSON, W . FRY AND D. SCHWEMMIN, J. Sen. Physiol., 38 (1955) 549. 2e E. F. GALE, J. Sen. Microbiol., I (1947) 53. a7 D. S. HOARE, J. Sen. Microbiol., i2 (1955) 534.
Received March 3Ist, 1958
COMPARATIVE BIOCHEMISTRY OF COLLAGEN SOME AMINO ACIDS AND CARBOHYDRATES* JEROME
G R O S S * * , B I R U T A D U M S H A AnD N O R M A G L A Z E R
Department o] Medicine, Haward Medical School and the Medical Services o/the Massachusetts General Hospital, Boston, Mass. (U.S.A .)
Phylogenetic studies of the collagen class of proteins by X-ray diffraction 1-4, covering many phyla including invertebrates, has revealed considerable uniformity with regard to molecular organization. Analytical studies have been more limited, particularly with regard to the invertebrates. Amino acid analyses have been reported for earthworm collagenS, e and incomplete data are available for spongins 7.s and the gorgonins 7. Recently a substance from the nematocysts of Hydra was found to contain 22 % hydroxyproline9.The carbohydrate contents of two spongins have been reported s. The four amino acids most characteristic of collagen are hydroxyproline, hydroxylysine, proline and glycine; the first two are unique (thus far) to collagen among animal proteins and the last two are present in unusually high concentrations. Tyrosine is of some interest because it is present in low concentrations in most collagen and there is much controversy as to whether it is part of the structure or a contaminant. We * T h i s is p u b l i c a t i o n No. 233 of t h e R o b e r t W. L o v e t t M e m o r i a l F o u n d a t i o n for t h e S t u d y of Crippling Disease, H a r v a r d Medical School a t t h e M a s s a c h u s e t t s General H o s p i t a l . Boston. This s t u d y w a s a i d e d b y g r a n t A9o (C5, 6 a n d 7) f r o m t h e N a t i o n a l I n s t i t u t e of A r t h r i t i s a n d Metabolic Diseases of t h e U n i t e d S t a t e s Public H e a l t h Service. ** T h i s w o r k w a s done d u r i n g t h e t e n u r e of a n E s t a b l i s h e d I n v e s t i g a t o r s h i p of t h e A m e r i c a u H e a r t Association.
Re/erences p. 297.
294
j . GROSS, B. DUMSHA, N. GLAZER
VOL. 3 0 (1958)
compare, in this paper, the content of these amino acids (except for hydroxylysine) and the sugars in the collagen, and gelatin derived therefrom, obtained from mammal, fish, echinoderm, coelenterate and sponge s. MATERIALS AND METHODS C o r i u m of n e w - b o r n calf, 13 y e a r - o l d s t e e r a n d t h e collagen isolated f r o m t h e l a t t e r b y e x h a u s t i v e e x t r a c t i o n in i o % NaC1, M[5 N a 2 H P O 4 a n d ~ s a t u r a t e d CaCOH)210 were u s e d as a baseline for c o m p a r i s o n . E l a s t o i d i n (shark, C. glaucus) a n d o v o k e r a t i n (skate, Rain battis) were k i n d l y p r o v i d e d b y R. FAURE-FREMIET. All o t h e r m a t e r i a l , e x c e p t t h e s p o n g e s (Florida) were o b t a i n e d fresh in M a s s a c h u s e t t s . T h e c o r i u m of fresh carp, Thyone a n d Metridium were m e c h a n i c a l l y freed of t h e e p i d e r m a l layers, w a s h e d t h o r o u g h l y w i t h w a t e r , dried in a c e t o n e a n d f r a g m e n t e d . I c h t h y o c o l w a s o b t a i n e d f r o m s a l i n e - w a s h e d c a r p s w i m - b l a d d e r t u n i c s b y e x t r a c t i o n w i t h o. 1 % acetic acid a n d p r e c i p i t a t i o n b y dialysis a g a i n s t o. 17 M NaC1. T h i s m a t e r i a l w a s redissolved a n d p r e c i p i t a t e d 3 X, dialyzed free of salt a n d lyophilized. E l a s t i n w a s p r e p a r e d f r o m t h e t u n i c r e s i d u e s b y r e p e a t e d e x t r a c t i o n in h o t i % acetic acid followed b y dialysis a n d lyophilization of t h e residue. Physalia float w a s w a s h e d in w a t e r a n d dried in acetone. L i m e d p r e p a r a t i o n s were e x t r a c t e d 4 × in t h e cold w i t h halfs a t u r a t e d c a l c i u m h y d r o x i d e a n d dialyzed a g a i n s t dilute HC1 before d r y i n g . G e l a t i n s were p r e p a r e d b y a u t o c l a v i n g o v e r n i g h t in w a t e r a t i5-1b, pressure, followed b y dialysis a n d lyophilization of t h e s u p e r n a t a n t fluids. T h e p r e p a r a t i o n , c h a r a c t e r i z a t i o n a n d a n a l y s i s of t h e t w o t y p e s of s p o n g i n f o u n d in fresh Spongia graminea were r e p o r t e d p r e v i o u s l y s. Colorimetric a n a l y s e s were m a d e for h y d r o x y p r o l i n e 11, proline TM, glycine x3, a n d t y r o s i n e 14, h e x o s a m i n e 15, h e x o s e (Orcinol m e t h o d ) xe, p e n t o s e x~, a n d m e t h y l p e n t o s e xs. H e x o s e s t a n d a r d w a s a m i x t u r e of e q u a l p a r t s of glu, gal, m a n , ara, fuc. H y d r o l y s a t e s for t h e first t h r e e a m i n o acids were p r e p a r e d b y h e a t i n g ~o-2o m g dried m a t e r i a l in I m l 6 N HC1 in a sealed g l a s s t u b e in a n oil b a t h a t 1384 for 3 h. All t h r e e a n a l y s e s were p e r f o r m e d on t h e s a m e h y d r o l y s a t e s . T y r o s i n e w a s m e a s u r e d in a n alkaline h y d r o l y s a t e . D e t e r m i n a t i o n s of h e x o s a m i n e were m a d e o n s a m p l e s h y d r o l y s e d for 16 h in 2 N HC1 a t IOO ° in sealed t u b e s . T h e a m i n o s u g a r s were s e p a r a t e d p r i o r to a n a l y s i s on a D o w e x - 5 o i o n - e x c h a n g e c o l u m n as described b y BoAs ~ . P a p e r c h r o m a t o g r a p h i c i d e n t i f i c a t i o n of h e x o s a m i n e s w a s a c c o m p l i s h e d b y t h e m e t h o d of STOFFYN AND JEASLOZ TM. T h e n e u t r a l a n d acid s u g a r fraction w a s o b t a i n e d b y r e f l u x i n g for 4 a n d 16 h in 2 N HC1 a t lOO% N e u t r a l a n d acid s u g a r s were s e p a r a t e d f r o m t h e a m i n o acicls b y w a t e r e l u t i o n f r o m a D o w e x - 5 o c o l u m n a n d c o n c e n t r a t e d in a s t r e a m of n i t r o g e n . T h e s u g a r s were c h r o m a t o g r a p h e d in t h e following t h r e e different s o l v e n t s y s t e m s , o n e - a n d t w o - d i m e n s i o n a l l y , as described p r e v i o u s l y s : B u t a n o l - p y r i d i n e - w a t e r , 3 : 2 : 1.5 ; e t h y l a c e t a t e - a c e t i c a c i d - w a t e r , 3 : i : 3 ; w a t e r - s a t u r a t e d phenol. S t a i n i n g w a s a c c o m p l i s h e d w i t h b o t h silver a n d aniline p h t h a l a t e . T y p i c a l s u g a r c h r o m a t o g r a m s of s p o n g i n h y d r o l y s a t e s were p r e s e n t e d elsewhere s.
RESULTS
Table I reports the results of the amino acid and sugar analyses on the different collagens and gelatins. In general, proline tended to be somewhat higher than hydroxyproline in the vertebrate collagens and equal or somewhat lower in the invertebrates. The lowest molar ratio of gly/hypro in collagen was found in Spongin A and the highest, in Metridiu~ (excluding ovokeratin). Tyrosine values were uniformly low, I ~o or less except for the carp swim-bladder tunic, the elaStin derived therefrom, carp scales, elastoidin and ovokeratin. It is evident from the large differences between the amino acid values for gelatin and the parent material of elastoidin, swim-bladder tunic and carp scales that a complex mixture exists in the parent substance. The tunic is a mixture of collagen and elastin. The latter, even after liming, contained a large amount of tyrosine. Elastoidin contains a non-collagenous material, rich in tyrosine, which is insoluble on autoclaving 2°. Re/erences p. 297.
26.2 29.1
Cow corium Cow collagen
30.2
2+6
27.0 17. 4 18.3 22.5 30.6 19.9 25.9 i 1.3 14-1 13.1
Carp c o r i u m gelatin
Carp s w i m bladder tunic
Carp s w i m bladder collagen Carp s w i m bladder elastin* Carp s w i m bladder elastin limed* Carp scales Carp scales gelatin S h a r k elastoidin S h a r k elastoidin gelatin Skate o v o k e r a t i n * Skate o v o k e r a t i n N a O H - t r e a t e d * Skate ovokeratin gelatin*
20. 4
18.8 19.2 2o.8
15.7
2o.5 2o. 5
Thyone corium gelatin
M e t r i d i u m corium* Melridium corium limed* Metridium corium gelatin*
Physalia float
Physalia float limed* Physalia float gelatin*
12.4
lO.2
6.2 6. 4
5.5
5.2 5.2 5.7
8.2
7.6
12.1 I.O 0.9 lO.2 I3.I 8.2 ii.i 2.3 2.4 3 .o
8.2
11.8
8.9
13. 7 14.3
ii.o
Hi,pro
7.2
7-4
5.5 5.6
5 .0
5"7 5.8 5.3
8. 5
9.6
13.1 6.2 9.9 11.9 14.9 lO.8 12.1 4.6 4-7 5 .8
11.6
14. 4
12.6
14. 9 16.1
i2.8
Pro
TABLE I
3.1
2.8
5.8 5-3
4.5
6.3 6.5 6. 4
4.4
4-5
3.9 30.4 35.4 3.8 4.1 4.3 4.1 8.3 lO.3 7 .6
5-3
4.6
4.8
3.3 3.6
3.7
o.2
1.8
o.2 o.9
0.5
I.O o.6 o. 7
o.6
I.I
0.02 o.9 o.6 o.2 0.08 0.03 o.o 3 o.2 0.o9 o.3
o.3
o.04
o. 4
0.2 o.oi
0.4
Mol ratio HexosGly/Hypro amine
4-9
11.o
4.8 6.o
5.7
8.9 5.1 6. 7
4.4
3.3
0.6 2.7 1. 4 o. 7 0. 7 o.7 1. 3 2.o I.O 2.7
i.o
o.8
I.O
0.8 o.5
1.5
Hex
o. 5
2.7
0.9 o.9
0.8
o.9 o.8 0.8
o.5
o.6
0. 5
0.I
0. 4
o. 13
o. 1 0.0 7 o.o 7
0.2
o.2 o.o7 o.4
o.2 0.07
o.o 5
O.1
O. I
Pent
•
glu, gal, m a n , ara, fuc, Ur. A & L glucosamine, g a l a c t o s a m i n e glu, gal
same
glu, gal, m a n , ara, rib ? * ** , fuc, r h a m ? ** * Ur. A & L, glucosamine, g a l a c t o s a m i n e
I glu,gal, man, ara, fuc, unkn, Ur. A & L ! glucosamine, g a l a c t o s a m i n e ~ glu, gal, ara, fuc, u n k n , Ur. A & L / glucosamine, g a l a c t o s a m i n e
glu, gal glu, gal
glu, gal
glu, gal, m a n
glu, gal, m a n , fuc rib glucosamine, galactosamine
glu, gal, m a n , ara, fuc, rib, Ur. A & L glucosamine, g a l a c t o s a m i n e
glu, gal
glu, gal, man, Ur. A & L** glucosamine, g a l a c t o s a m i n e
Sugars identified by paper chromatography
COLLAGENS AND GELATINS
Crrams/xoo grams dry ash-/fee weight
AND INVERTEBRATE
*** Spot faint or o b s e r v e d in o n l y one of t h r e e s o l v e n t systems.
o.6
0.9
1.2 1.2
1-5
1.5 1.2 o.9
1. 5
1.7
0. 3 6.3 6.9 1. 3 0. 7 6. 5 1.9 8.o 9.o 5.7
1. 4
o. 5
I .o
0.9 o. 4
1. 3
Tyr
OF VERTEBRATE
** Uronic acid a n d lactone.
22.4
Spongin B
* Ash d e t e r m i n a t i o n s n o t done.
16. 4
Spongin A
PORIFERA
COELENTERATE
19.8
Thyone corium
ECHINODERM
24.8
Carp c o r i u m
FISH
23.2
Calf c o r i u m
MAMMAL
Gly
PARTIAL COMPOSITION OF A VARIETY
O
O
O m
©
q~ O
296
J. GROSS, B. DUMSHA, N. GLAZER
VOL. 30 (1958)
Ovokeratin, a secreted collagen21, 3 (based on a weak, poorly oriented collagen wide-angle X-ray diffraction pattern), is most likely a complex mixture of materials. Little separation from its high tyrosine moiety was accomplished by autoclaving, as compared with elastoidin. The maximum total-sugar content for the vertebrate connective tissues was 2 % in the calf corium (excluding ovokeratin, 2.6 %) and the lowest value, 0.6 %, was found in the highly purified steer-hide collagen. The invertebrate connective tissues, in contrast, ranged from a high of 15.5 % in Spongin A to a low of 5.o % in Thyonecorium. Hexose in all cases accounted for the largest fraction of sugar. Purification or gelatinization drastically reduced the hexosamine content in the vertebrate tissues and was less effective in this regard with the invertebrates. Physalia float gelatin actually contained more hexosamine than did the parent tissue. Hexose, on the other hand, was hot greatly reduced in the gelatins, and, in fact, Thyoneand Physalia gelatin contained more hexose than did the parent tissues. A remarkably large amount of methyl pentose, 1.2 % identified as fucose, was found in Thyone skin. It is yet to be determined whether or not the carbohydrate is firmly bonded to the protein. The elastin of the carp swim-bladder carried a large amount of the total swimbladder sugar with it. However, this might be explained by the insolubilization of negatively charged carbohydrates by complexing with the proteins in the acid medium used for isolation. Liming reduced the figure to half. Any more intimate structural association is yet to be demonstrated. All the samples examined chromatographically revealed glucose, galactose, glucosamine and galactosamine. Fucose was found in all except the calf corium, swimbladder elastin, carp scales and elastoidin and was a heavily accentuated spot on the Thyone chromatogram. Mannose was found in all preparations examined except carp scales and shark elastoidin. Arabinose, previously reported in sponge collagens s, was also present in Physalia float and, in much smaller amounts, in Thyone corium. Neither ribose nor desoxyribose were detected in the invertebrate preparations. A spot with Re value of rhamnose appeared in the hydrolysates of Physaliafloat in two of the three solvent systems. An unidentified reducing spot was observed on the chromatograms of Thyone. DISCUSSION
All the tissues examined have yielded typical collagen wide-angle X-ray diffraction patterns. Electron micrographs taken in this laboratory have revealed the characteristic 6oo-7oo/~ axial period in the fibers of all these animal tissues with the exception of the swim-bladder elastin, ovokeratin, Physaliafloat and Metridium body wall. The last two are composed of masses of fibrils of the order of 2oo A in diameter with a faint, as yet unsatisfactorily resolved axial periodicity. Glycine and proline values for the swim-bladder elastin are lower than for mammalian elastin and the tyrosine is higher; hydroxyproline content is similar. The relative proportions of glycine, hydroxyproline and proline are the same ordcr of magnitude for all the collagens reported here, with the exception of ovokeratin, and seem to be consistent with the requirements of current structural models of the collagen molecule. More complete amino acid analyses will be reported at a later date. Re/erences p. 297.
voL. 3 0 (1958)
COMPARATIVE BIOCHEMISTRY OF COLLAGEN
297
The large variety of sugars associated with the collagens and gelatins suggest that if the carbohydrate is in polysaccharide form, there is probably more than one molecular species present. Whether or not the carbohydrate moiety is an intrinsic part of the collagen molecule or became bonded to the fibril as an innocent bystander during fibrogenesis is an open question. SUMMARY H y d r o x y p r o l i n e , proline, glycine, t y r o s i n e a n d t h e c a r b o h y d r a t e c o m p o s i t i o n of eleven n a t i v e a n d limed collagens a n d derived gelatins f r o m a m a m m a l , fish, e c h i n o d e r m , t w o c o e l e n t e r a t e s a n d a poriferan p l u s t h e c o m p o s i t i o n of a fish elastin are reported. REFERENCES 1 W. T. ASTBURY, J. Intern. Soc. Leather Trade Chemists, 24 (194o) 69,. 2 M. H. MARKS, R. S. BEAR AND C. H. BLAKE, J. Exptl. Zool., 111 (1949) 55. a R. S. BEAR, Advances in Protein Chem., 7 (195 °) 69. 4 K. M. RODALL, Symposia Soc. Exptl. Biol., 9 (1955) 49. 5 M. R. WATSON, Biochem. J., 68 (1958) 416. 6 L. SINGLETON, Biochim. Biophys. Acta, 24 (1957) 67. 7 j . R o c h e AND R. MICHEL, Advances in Protein Chem., 6 (1951) 253. a j . GRoss, Z. SOKAL AND M. ROUGVlE, J. Histochem. and Cytochem., 4 (1956) 227. 9 H. M. LENnOFF, E. S. KLINE AND R. HURLEY, Biochim. Biophys. Acta, 26 (1957) 2o4. 10 F. O. SCHMITX, J. GROSS AND J. H. HIG~IEERGEa, Symposia Soc. Exptl. Biol., 9 (1955) 148. 11 R. E. NEUMANN AND M. A. LOGAN, J. Biol. Chem., 184 (195o) 299. 12 W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. 11 H. N. CHRISTENSEN, T. R. RIGGS AND N. E. RAY, Anal. Chem., 23 (1951) 1521. 14 F. W. BERNHARDT, J. Biol. Chem., 123 (1938) x. 15 N. F. BOAS, J. Biol. Chem., 204 (I953) 553. 16 R. FRIEDMANN, Biochem. J., 44 (1949) 117. aT W. MEJEAUM, Z. physio !. Chem., 258 (1939) 117. 18 Z. DIscHE AND L. B. SX~EXTLES, J. Biol. Chem., 175 (1948) 595. 19 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. 80 j . G R o s s AND B. DUMSI-IA, Biochim. Biophys. Acta, 28 (1958) 268. 21 G. CBAMPETIER AND E. FAURE-FEEMmT, J. cairn, phys., 34 (1938) 197.
Received April 9th, 1958
Addendum (added in proof, receive~l S e p t e m b e r i6th) Sialic acid d e t e r m i n a t i o n s (L. SVENNERHOLM, Acta Chim. Stand., I2 (1958) 547) p e r f o r m e d b y Dr. E. EYLAR of this u n i t on all t h e collagens r e p o r t e d here revealed less t h a n o. i % in a l l e x c e p t calf skin, 0.2 % ; s w i m - b l a d d e r tunic, o.15 % ; a n d s w i m - b l a d d e r elastin, o.3 %.
VOL. 30 (1958)
293
* J. B. LEATHES AND H. S. RAPER, The Fats, L o n g m a n s , G r e e n & Co., L o n d o n , 1925, p. 58. x0 W. R. BLOOR, J. Biol. Chem., 17o (1947) 671. 11 R. B. ROBERTS, D. B. COWIE, P. H. ABELSON, E. T. BOLTON AND R. J. BRITTEN, Studies o[ Biosynthesis in Escherichia cull, Carnegie I n s t i t u t i o n of W a s h i n g t o n , 1955. 12 S. DAGLEY AND J. SYKES, p e r s o n a l c o m m u n i c a t i o n . 13 S. DAGLEY, E. A. DAWES AND G. A. MORRISON, J. Sen. Microbiol., 4 (195 °) 437. i4 D. E. HUGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 15 L. H. STICKLAND,Biochem. J., 64 (1956) 498. is E. A. DAWES AND W. H. HOLMS, Biochim. Biophys. Acts, 29 (1958) 82. 17 W . SAKAMI,Handbook o] Isotope Tracer Methods, W e s t e r n R e s e r v e U n i v e r s i t y , Cleveland, Ohio. x955. is p. H. KOPPER, J. Bacteriol., 63 (1952) 639. x9 p. H. KOPPER, J. Bacteriol., 67 (1954) 5o7 • 20 D. JERCHEL AND H. FISCHEr, Ann., 563 (1949) 2oo. 21 F. C. NORRIS, J. j . R. CAMPBELL AND P. W. NEY, Can. J. Research, C27 ([949) 15721I j . LASCELLES, Biochem. J . , 62 (1956) 78. ~8 R. COOPER, Biockem. J., 63 (1956) 2 5 P . H. K. SCHACHMAN, A. B. PARDEE AND R. Y. STANIER, Arch. Biockem. Biophys., 38 (1952) 245. H. HALVORSON, W . FRY AND D. SCHWEMMIN, J. Sen. Physiol., 38 (1955) 549. 2e E. F. GALE, J. Sen. Microbiol., I (1947) 53. a7 D. S. HOARE, J. Sen. Microbiol., i2 (1955) 534.
Received March 3Ist, 1958
COMPARATIVE BIOCHEMISTRY OF COLLAGEN SOME AMINO ACIDS AND CARBOHYDRATES* JEROME
G R O S S * * , B I R U T A D U M S H A AnD N O R M A G L A Z E R
Department o] Medicine, Haward Medical School and the Medical Services o/the Massachusetts General Hospital, Boston, Mass. (U.S.A .)
Phylogenetic studies of the collagen class of proteins by X-ray diffraction 1-4, covering many phyla including invertebrates, has revealed considerable uniformity with regard to molecular organization. Analytical studies have been more limited, particularly with regard to the invertebrates. Amino acid analyses have been reported for earthworm collagenS, e and incomplete data are available for spongins 7.s and the gorgonins 7. Recently a substance from the nematocysts of Hydra was found to contain 22 % hydroxyproline9.The carbohydrate contents of two spongins have been reported s. The four amino acids most characteristic of collagen are hydroxyproline, hydroxylysine, proline and glycine; the first two are unique (thus far) to collagen among animal proteins and the last two are present in unusually high concentrations. Tyrosine is of some interest because it is present in low concentrations in most collagen and there is much controversy as to whether it is part of the structure or a contaminant. We * T h i s is p u b l i c a t i o n No. 233 of t h e R o b e r t W. L o v e t t M e m o r i a l F o u n d a t i o n for t h e S t u d y of Crippling Disease, H a r v a r d Medical School a t t h e M a s s a c h u s e t t s General H o s p i t a l . Boston. This s t u d y w a s a i d e d b y g r a n t A9o (C5, 6 a n d 7) f r o m t h e N a t i o n a l I n s t i t u t e of A r t h r i t i s a n d Metabolic Diseases of t h e U n i t e d S t a t e s Public H e a l t h Service. ** T h i s w o r k w a s done d u r i n g t h e t e n u r e of a n E s t a b l i s h e d I n v e s t i g a t o r s h i p of t h e A m e r i c a u H e a r t Association.
Re/erences p. 297.
294
j . GROSS, B. DUMSHA, N. GLAZER
VOL. 3 0 (1958)
compare, in this paper, the content of these amino acids (except for hydroxylysine) and the sugars in the collagen, and gelatin derived therefrom, obtained from mammal, fish, echinoderm, coelenterate and sponge s. MATERIALS AND METHODS C o r i u m of n e w - b o r n calf, 13 y e a r - o l d s t e e r a n d t h e collagen isolated f r o m t h e l a t t e r b y e x h a u s t i v e e x t r a c t i o n in i o % NaC1, M[5 N a 2 H P O 4 a n d ~ s a t u r a t e d CaCOH)210 were u s e d as a baseline for c o m p a r i s o n . E l a s t o i d i n (shark, C. glaucus) a n d o v o k e r a t i n (skate, Rain battis) were k i n d l y p r o v i d e d b y R. FAURE-FREMIET. All o t h e r m a t e r i a l , e x c e p t t h e s p o n g e s (Florida) were o b t a i n e d fresh in M a s s a c h u s e t t s . T h e c o r i u m of fresh carp, Thyone a n d Metridium were m e c h a n i c a l l y freed of t h e e p i d e r m a l layers, w a s h e d t h o r o u g h l y w i t h w a t e r , dried in a c e t o n e a n d f r a g m e n t e d . I c h t h y o c o l w a s o b t a i n e d f r o m s a l i n e - w a s h e d c a r p s w i m - b l a d d e r t u n i c s b y e x t r a c t i o n w i t h o. 1 % acetic acid a n d p r e c i p i t a t i o n b y dialysis a g a i n s t o. 17 M NaC1. T h i s m a t e r i a l w a s redissolved a n d p r e c i p i t a t e d 3 X, dialyzed free of salt a n d lyophilized. E l a s t i n w a s p r e p a r e d f r o m t h e t u n i c r e s i d u e s b y r e p e a t e d e x t r a c t i o n in h o t i % acetic acid followed b y dialysis a n d lyophilization of t h e residue. Physalia float w a s w a s h e d in w a t e r a n d dried in acetone. L i m e d p r e p a r a t i o n s were e x t r a c t e d 4 × in t h e cold w i t h halfs a t u r a t e d c a l c i u m h y d r o x i d e a n d dialyzed a g a i n s t dilute HC1 before d r y i n g . G e l a t i n s were p r e p a r e d b y a u t o c l a v i n g o v e r n i g h t in w a t e r a t i5-1b, pressure, followed b y dialysis a n d lyophilization of t h e s u p e r n a t a n t fluids. T h e p r e p a r a t i o n , c h a r a c t e r i z a t i o n a n d a n a l y s i s of t h e t w o t y p e s of s p o n g i n f o u n d in fresh Spongia graminea were r e p o r t e d p r e v i o u s l y s. Colorimetric a n a l y s e s were m a d e for h y d r o x y p r o l i n e 11, proline TM, glycine x3, a n d t y r o s i n e 14, h e x o s a m i n e 15, h e x o s e (Orcinol m e t h o d ) xe, p e n t o s e x~, a n d m e t h y l p e n t o s e xs. H e x o s e s t a n d a r d w a s a m i x t u r e of e q u a l p a r t s of glu, gal, m a n , ara, fuc. H y d r o l y s a t e s for t h e first t h r e e a m i n o acids were p r e p a r e d b y h e a t i n g ~o-2o m g dried m a t e r i a l in I m l 6 N HC1 in a sealed g l a s s t u b e in a n oil b a t h a t 1384 for 3 h. All t h r e e a n a l y s e s were p e r f o r m e d on t h e s a m e h y d r o l y s a t e s . T y r o s i n e w a s m e a s u r e d in a n alkaline h y d r o l y s a t e . D e t e r m i n a t i o n s of h e x o s a m i n e were m a d e o n s a m p l e s h y d r o l y s e d for 16 h in 2 N HC1 a t IOO ° in sealed t u b e s . T h e a m i n o s u g a r s were s e p a r a t e d p r i o r to a n a l y s i s on a D o w e x - 5 o i o n - e x c h a n g e c o l u m n as described b y BoAs ~ . P a p e r c h r o m a t o g r a p h i c i d e n t i f i c a t i o n of h e x o s a m i n e s w a s a c c o m p l i s h e d b y t h e m e t h o d of STOFFYN AND JEASLOZ TM. T h e n e u t r a l a n d acid s u g a r fraction w a s o b t a i n e d b y r e f l u x i n g for 4 a n d 16 h in 2 N HC1 a t lOO% N e u t r a l a n d acid s u g a r s were s e p a r a t e d f r o m t h e a m i n o acicls b y w a t e r e l u t i o n f r o m a D o w e x - 5 o c o l u m n a n d c o n c e n t r a t e d in a s t r e a m of n i t r o g e n . T h e s u g a r s were c h r o m a t o g r a p h e d in t h e following t h r e e different s o l v e n t s y s t e m s , o n e - a n d t w o - d i m e n s i o n a l l y , as described p r e v i o u s l y s : B u t a n o l - p y r i d i n e - w a t e r , 3 : 2 : 1.5 ; e t h y l a c e t a t e - a c e t i c a c i d - w a t e r , 3 : i : 3 ; w a t e r - s a t u r a t e d phenol. S t a i n i n g w a s a c c o m p l i s h e d w i t h b o t h silver a n d aniline p h t h a l a t e . T y p i c a l s u g a r c h r o m a t o g r a m s of s p o n g i n h y d r o l y s a t e s were p r e s e n t e d elsewhere s.
RESULTS
Table I reports the results of the amino acid and sugar analyses on the different collagens and gelatins. In general, proline tended to be somewhat higher than hydroxyproline in the vertebrate collagens and equal or somewhat lower in the invertebrates. The lowest molar ratio of gly/hypro in collagen was found in Spongin A and the highest, in Metridiu~ (excluding ovokeratin). Tyrosine values were uniformly low, I ~o or less except for the carp swim-bladder tunic, the elaStin derived therefrom, carp scales, elastoidin and ovokeratin. It is evident from the large differences between the amino acid values for gelatin and the parent material of elastoidin, swim-bladder tunic and carp scales that a complex mixture exists in the parent substance. The tunic is a mixture of collagen and elastin. The latter, even after liming, contained a large amount of tyrosine. Elastoidin contains a non-collagenous material, rich in tyrosine, which is insoluble on autoclaving 2°. Re/erences p. 297.
26.2 29.1
Cow corium Cow collagen
30.2
2+6
27.0 17. 4 18.3 22.5 30.6 19.9 25.9 i 1.3 14-1 13.1
Carp c o r i u m gelatin
Carp s w i m bladder tunic
Carp s w i m bladder collagen Carp s w i m bladder elastin* Carp s w i m bladder elastin limed* Carp scales Carp scales gelatin S h a r k elastoidin S h a r k elastoidin gelatin Skate o v o k e r a t i n * Skate o v o k e r a t i n N a O H - t r e a t e d * Skate ovokeratin gelatin*
20. 4
18.8 19.2 2o.8
15.7
2o.5 2o. 5
Thyone corium gelatin
M e t r i d i u m corium* Melridium corium limed* Metridium corium gelatin*
Physalia float
Physalia float limed* Physalia float gelatin*
12.4
lO.2
6.2 6. 4
5.5
5.2 5.2 5.7
8.2
7.6
12.1 I.O 0.9 lO.2 I3.I 8.2 ii.i 2.3 2.4 3 .o
8.2
11.8
8.9
13. 7 14.3
ii.o
Hi,pro
7.2
7-4
5.5 5.6
5 .0
5"7 5.8 5.3
8. 5
9.6
13.1 6.2 9.9 11.9 14.9 lO.8 12.1 4.6 4-7 5 .8
11.6
14. 4
12.6
14. 9 16.1
i2.8
Pro
TABLE I
3.1
2.8
5.8 5-3
4.5
6.3 6.5 6. 4
4.4
4-5
3.9 30.4 35.4 3.8 4.1 4.3 4.1 8.3 lO.3 7 .6
5-3
4.6
4.8
3.3 3.6
3.7
o.2
1.8
o.2 o.9
0.5
I.O o.6 o. 7
o.6
I.I
0.02 o.9 o.6 o.2 0.08 0.03 o.o 3 o.2 0.o9 o.3
o.3
o.04
o. 4
0.2 o.oi
0.4
Mol ratio HexosGly/Hypro amine
4-9
11.o
4.8 6.o
5.7
8.9 5.1 6. 7
4.4
3.3
0.6 2.7 1. 4 o. 7 0. 7 o.7 1. 3 2.o I.O 2.7
i.o
o.8
I.O
0.8 o.5
1.5
Hex
o. 5
2.7
0.9 o.9
0.8
o.9 o.8 0.8
o.5
o.6
0. 5
0.I
0. 4
o. 13
o. 1 0.0 7 o.o 7
0.2
o.2 o.o7 o.4
o.2 0.07
o.o 5
O.1
O. I
Pent
•
glu, gal, m a n , ara, fuc, Ur. A & L glucosamine, g a l a c t o s a m i n e glu, gal
same
glu, gal, m a n , ara, rib ? * ** , fuc, r h a m ? ** * Ur. A & L, glucosamine, g a l a c t o s a m i n e
I glu,gal, man, ara, fuc, unkn, Ur. A & L ! glucosamine, g a l a c t o s a m i n e ~ glu, gal, ara, fuc, u n k n , Ur. A & L / glucosamine, g a l a c t o s a m i n e
glu, gal glu, gal
glu, gal
glu, gal, m a n
glu, gal, m a n , fuc rib glucosamine, galactosamine
glu, gal, m a n , ara, fuc, rib, Ur. A & L glucosamine, g a l a c t o s a m i n e
glu, gal
glu, gal, man, Ur. A & L** glucosamine, g a l a c t o s a m i n e
Sugars identified by paper chromatography
COLLAGENS AND GELATINS
Crrams/xoo grams dry ash-/fee weight
AND INVERTEBRATE
*** Spot faint or o b s e r v e d in o n l y one of t h r e e s o l v e n t systems.
o.6
0.9
1.2 1.2
1-5
1.5 1.2 o.9
1. 5
1.7
0. 3 6.3 6.9 1. 3 0. 7 6. 5 1.9 8.o 9.o 5.7
1. 4
o. 5
I .o
0.9 o. 4
1. 3
Tyr
OF VERTEBRATE
** Uronic acid a n d lactone.
22.4
Spongin B
* Ash d e t e r m i n a t i o n s n o t done.
16. 4
Spongin A
PORIFERA
COELENTERATE
19.8
Thyone corium
ECHINODERM
24.8
Carp c o r i u m
FISH
23.2
Calf c o r i u m
MAMMAL
Gly
PARTIAL COMPOSITION OF A VARIETY
O
O
O m
©
q~ O
296
J. GROSS, B. DUMSHA, N. GLAZER
VOL. 30 (1958)
Ovokeratin, a secreted collagen21, 3 (based on a weak, poorly oriented collagen wide-angle X-ray diffraction pattern), is most likely a complex mixture of materials. Little separation from its high tyrosine moiety was accomplished by autoclaving, as compared with elastoidin. The maximum total-sugar content for the vertebrate connective tissues was 2 % in the calf corium (excluding ovokeratin, 2.6 %) and the lowest value, 0.6 %, was found in the highly purified steer-hide collagen. The invertebrate connective tissues, in contrast, ranged from a high of 15.5 % in Spongin A to a low of 5.o % in Thyonecorium. Hexose in all cases accounted for the largest fraction of sugar. Purification or gelatinization drastically reduced the hexosamine content in the vertebrate tissues and was less effective in this regard with the invertebrates. Physalia float gelatin actually contained more hexosamine than did the parent tissue. Hexose, on the other hand, was hot greatly reduced in the gelatins, and, in fact, Thyoneand Physalia gelatin contained more hexose than did the parent tissues. A remarkably large amount of methyl pentose, 1.2 % identified as fucose, was found in Thyone skin. It is yet to be determined whether or not the carbohydrate is firmly bonded to the protein. The elastin of the carp swim-bladder carried a large amount of the total swimbladder sugar with it. However, this might be explained by the insolubilization of negatively charged carbohydrates by complexing with the proteins in the acid medium used for isolation. Liming reduced the figure to half. Any more intimate structural association is yet to be demonstrated. All the samples examined chromatographically revealed glucose, galactose, glucosamine and galactosamine. Fucose was found in all except the calf corium, swimbladder elastin, carp scales and elastoidin and was a heavily accentuated spot on the Thyone chromatogram. Mannose was found in all preparations examined except carp scales and shark elastoidin. Arabinose, previously reported in sponge collagens s, was also present in Physalia float and, in much smaller amounts, in Thyone corium. Neither ribose nor desoxyribose were detected in the invertebrate preparations. A spot with Re value of rhamnose appeared in the hydrolysates of Physaliafloat in two of the three solvent systems. An unidentified reducing spot was observed on the chromatograms of Thyone. DISCUSSION
All the tissues examined have yielded typical collagen wide-angle X-ray diffraction patterns. Electron micrographs taken in this laboratory have revealed the characteristic 6oo-7oo/~ axial period in the fibers of all these animal tissues with the exception of the swim-bladder elastin, ovokeratin, Physaliafloat and Metridium body wall. The last two are composed of masses of fibrils of the order of 2oo A in diameter with a faint, as yet unsatisfactorily resolved axial periodicity. Glycine and proline values for the swim-bladder elastin are lower than for mammalian elastin and the tyrosine is higher; hydroxyproline content is similar. The relative proportions of glycine, hydroxyproline and proline are the same ordcr of magnitude for all the collagens reported here, with the exception of ovokeratin, and seem to be consistent with the requirements of current structural models of the collagen molecule. More complete amino acid analyses will be reported at a later date. Re/erences p. 297.
voL. 3 0 (1958)
COMPARATIVE BIOCHEMISTRY OF COLLAGEN
297
The large variety of sugars associated with the collagens and gelatins suggest that if the carbohydrate is in polysaccharide form, there is probably more than one molecular species present. Whether or not the carbohydrate moiety is an intrinsic part of the collagen molecule or became bonded to the fibril as an innocent bystander during fibrogenesis is an open question. SUMMARY H y d r o x y p r o l i n e , proline, glycine, t y r o s i n e a n d t h e c a r b o h y d r a t e c o m p o s i t i o n of eleven n a t i v e a n d limed collagens a n d derived gelatins f r o m a m a m m a l , fish, e c h i n o d e r m , t w o c o e l e n t e r a t e s a n d a poriferan p l u s t h e c o m p o s i t i o n of a fish elastin are reported. REFERENCES 1 W. T. ASTBURY, J. Intern. Soc. Leather Trade Chemists, 24 (194o) 69,. 2 M. H. MARKS, R. S. BEAR AND C. H. BLAKE, J. Exptl. Zool., 111 (1949) 55. a R. S. BEAR, Advances in Protein Chem., 7 (195 °) 69. 4 K. M. RODALL, Symposia Soc. Exptl. Biol., 9 (1955) 49. 5 M. R. WATSON, Biochem. J., 68 (1958) 416. 6 L. SINGLETON, Biochim. Biophys. Acta, 24 (1957) 67. 7 j . R o c h e AND R. MICHEL, Advances in Protein Chem., 6 (1951) 253. a j . GRoss, Z. SOKAL AND M. ROUGVlE, J. Histochem. and Cytochem., 4 (1956) 227. 9 H. M. LENnOFF, E. S. KLINE AND R. HURLEY, Biochim. Biophys. Acta, 26 (1957) 2o4. 10 F. O. SCHMITX, J. GROSS AND J. H. HIG~IEERGEa, Symposia Soc. Exptl. Biol., 9 (1955) 148. 11 R. E. NEUMANN AND M. A. LOGAN, J. Biol. Chem., 184 (195o) 299. 12 W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. 11 H. N. CHRISTENSEN, T. R. RIGGS AND N. E. RAY, Anal. Chem., 23 (1951) 1521. 14 F. W. BERNHARDT, J. Biol. Chem., 123 (1938) x. 15 N. F. BOAS, J. Biol. Chem., 204 (I953) 553. 16 R. FRIEDMANN, Biochem. J., 44 (1949) 117. aT W. MEJEAUM, Z. physio !. Chem., 258 (1939) 117. 18 Z. DIscHE AND L. B. SX~EXTLES, J. Biol. Chem., 175 (1948) 595. 19 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. 80 j . G R o s s AND B. DUMSI-IA, Biochim. Biophys. Acta, 28 (1958) 268. 21 G. CBAMPETIER AND E. FAURE-FEEMmT, J. cairn, phys., 34 (1938) 197.
Received April 9th, 1958
Addendum (added in proof, receive~l S e p t e m b e r i6th) Sialic acid d e t e r m i n a t i o n s (L. SVENNERHOLM, Acta Chim. Stand., I2 (1958) 547) p e r f o r m e d b y Dr. E. EYLAR of this u n i t on all t h e collagens r e p o r t e d here revealed less t h a n o. i % in a l l e x c e p t calf skin, 0.2 % ; s w i m - b l a d d e r tunic, o.15 % ; a n d s w i m - b l a d d e r elastin, o.3 %.