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Effects of ultraviolet irradiation on native and telopeptide-poor collagen
672
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35807 EFFECTS OF U L T R A V I O L E T IRRADIATION ON NATIVE AND T E L O P E P T I D E - P O O R COLLAGEN
TERUO MIYATA, T A K E S H I SOHDE, ALBERT L. R U B I N AND K U R T H. STENZEL
Japan Leather Co., Ltd., Tokyo (Japan), and the Rogosin Laboratories, Departments of Biochemistry and Surgery, The New York Hospital-Cornell Medical Center, New York, N.Y. (U.S.A.) (Received October 23rd, 197 o)
SUMMARY
Effects of ultraviolet light on physical, chemical and immunologic properties of acid-soluble and enzyme (proctase)-treated collagen were studied. Ultraviolet irradiation in air caused an initial increase in viscosity of both collagen preparations, rapidly followed by a loss of viscosity and a loss of negative optical rotation. Ultraviolet irradiation in nitrogen, however, led to a rapid increase in viscosity until a gel formed. With prolonged irradiation, this gel also depolymerized. Enzyme-treated collagen was less sensitive to the effects of ultraviolet irradiation than was acidsoluble collagen. Immunologic reactivity of enzyme-treated collagen was rapidly lost with short exposures to ultraviolet irradiation. Ultraviolet irradiation also inhibited fiber formation. These results suggest that photochemical modifications occur at telopeptide regions or remaining aromatic residues of enzyme-treated collagen during early stages of irradiation. The transition temperature of the melting curve of collagen became broad but there was no loss of negative optical rotation, suggesting that scission of collagen molecules into shorter fragments which retain helical configurations occurred with longer periods of irradiation. Splitting of the polypeptide chain probably occurs between nitrogen and the a-carbon.
INTRODUCTION
We have attempted to use animal collagen for a variety of biomedical applications. Ultraviolet irradiation appears to be an ideal means of cross-linking and restructuring enzyme solubilized collagen, since it introduces no possibly toxic substituents, and is simple and effective. Several reports indicate that the structure of collagen is modified by ultraviolet irradiation. One effect of ultraviolet irradiation is reported to be a scission of primary collagen polypeptide chains l-a, while other effects appear to be photopolymerization of collagen and fibril formation 4,5. The primary chemical phenomena occurring during the initial stages of these modifications of collagen are thought to Biochim. Biophys. Acta, 229 (1971) 672-680
ULTRAVIOLET IRRADIATION OF COLLAGEN
673
be photochemical reactions involving tyrosine and phenylalanine. The telopeptide or end-chain regions of collagen are relatively rich in aromatic residues e, and also contain important antigenic determinants of collagen. We have determined the effects of ultraviolet irradiation on chemical, physical and immunologic properties of native (acid-soluble) and proctase-treated (telopeptide poor) collagen in both air and nitrogen atmospheres.
MATERIALS AND METHODS
Preparation of acid-soluble collagen Acid-soluble collagen was extracted from calf skin with o.15 M citrate buffer (pH 3.6) and purified by reprecipitation by dialysis against 0.02 M Na2HP04. The reconstituted fiber was then washed with water, dissolved in 0.05% acetic acid and dialyzed against 0.05 % acetic acid.
Proctase treatment of acid-soluble collagen Acid-soluble collagen in 0.05% acetic acid was treated with proctase A* (I/2OO) at 20 ° for 24 h, then dialyzed first against 0.02 M Na,HPO 4 and then against a borax buffer solution (0.037 M Na2C03-o.oi2 M Na,B407, pH IO.O) at 20 ° to inactivate the enzyme. The reconstituted fiber was then washed with water.
Ultraviolet irradiation Collagen solutions (O.lO9% in 0.05% acetic acid) were irradiated at 4 ° with a 4-W ultraviolet lamp (Sankyo Electric Co.) irradiating primarily at 2,537 A. Solutions were placed in IOO ml fused quartz flasks at a distance of io cm from the lamp. Irradiation was carried out either in the presence of air or in oxygen-free nitrogen gas. Nitrogen was passed through the collagen solutions for 30 min before, and then continued during the entire period of irradiation.
Optical rotation Optical rotation was measured at 20 4- o.i ° in a 2o-cm water-iacketed tube in a Rudolf (Model 8o) photoelectric polarimeter equipped with an oscillating polarizer pi'ism. Readings were taken at 589 m/~. Melting curves were determined by measuring optical rotation of collagen solutions as a function of temperature. The temperature of the solutions was raised at a rate of I°/IO min.
Viscosity Viscosities were measured at 20 4 - o . o i ° in an Ostwald-Fenske viscometer having a water flow time of 200 sec at 20 ° and a velocity gradient of I,OOO sec -1.
* Proctase A is an acidic proteolytic enzyme obtained from the incubation b r o t h of A spergillus niger var. macrosporous, has a p H o p t i m u m of 3.0 and was kindly supplied b y Dr. Koaze of Meiji Seika Kaisha, Ltd.
Biochim. Biophys. Acta, 229 (1971) 672-680
674
T. MIYATAet al.
Amino acid analysis Amino acid compositions were determined in a Hitachi amino acid analyzer Model KLA-2 following hydrolysis with 6 M HC1 under vacuum at i i o ° for 16 h.
Ninhydrin reactions Colorimetric determinations of irradiated collagen solutions with ninhydrin were done using a leucine standard.
Fibril formation Collagen solutions (0.1%) in 0.05% acetic acid were preheated at 3 °° for 2 h and mixed with an equal volume of phosphate buffer (0.o4 M Na~HPO4-o. 3 M KH2PO4-o.27 M NaC1, pH 8.5) preheated to 37 °. After mixing, extinction of collagen solutions at 4o0 m# was measured as a function of time at'37 ° in a Beckman DUtype spectrophotometer. The final mixture had a pH of 7.2, an ionic strength of o.155 and a collagen concentration of 0.o50/0 .
Complement fixation Complement fixation studies were done by the method of WASSERMAN AND LEVINE8 utilizing antibodies produced in rabbits to proctase-treated acid-soluble collagen.
RESULTS
Effects of ultraviolet irradiation on physical properties of collagen Optical rotation of acid-soluble and proctase-treated acid-soluble collagen was measured as a function of time of ultraviolet irradiation in both air and nitrogen atmospheres and results are shown in Fig. I. The specific rotation of acid-soluble collagen decreased more rapidly than that of proctase-treated acid-soluble collagen when irradiated either in air or in nitrogen. Irradiation in air caused more rapid degradation of the collagen triple helix, as indicated by the sharp decrease in specific rotation after 5 h irradiation for acid-soluble collagen and after IO h irradiation for proctase-treated acid-soluble collagen. Reduced viscosity of acid-soluble collagen and proctase-treated acid-soluble collagen irradiated in both air and nitrogen are depicted in Fig. 2. Irradiation in nitrogen caused a rapid increase in viscosity and gels were formed after 2 h irradiation of acid-soluble collagen and after 4 h irradiation of proctase-treated acid-soluble collagen. Acid-soluble collagen gels melted after 17 h irradiation and proctase-treated acid-soluble collagen gels melted after IO h irradiation. Thus, during early stages of irradiation, cross-linking reactions between collagen molecules appear to predominate, whereas with prolonged irradiation, degradation occurs. Irradiation in air, rather than in nitrogen, results in a slower increase in viscosity of both collagen preparations, but gels do not form and viscosity begins to decrease after 4 h irradiation. Thus, depolymerization, rather than cross-linking, seems to predominate when irradiation is done in air. Melting temperature decreases as irradiation time increases, as illustrated in Fig. 3. No significant differences between melting curves of untreated and proctaseBiochim. Biophys. Acta, 229 (I97 I) 672-68o
675
ULTRAVIOLET IRRADIATION OF COLLAGEN
40@ 60
'
20C
I00
0
I0
0
20
30
40
50
lrradlatton tlme ihours)
l0
ZO 30 Irradlatlon tlme thours)
40
Fig. I. Effect of ultraviolet irradiation on optical rotation of collagen solutions. 0 - - 0 , acidsoluble collagen irradiated in air; 0 - - - 0 , acid-soluble collagen irradiated in nitrogen; A - - A , proctase-treated acid-soluble collagen irradiated in air; A - - A , proctase-treated acid-soluble collagen irradiated in nitrogen. Collagen concn., o.Io9%; temp., 2o°; solvent, o.o 5 % acetic acid. Fig. 2. Effect of ultraviolet irradiation on reduced viscosity of collagen solutions. O - - O , acidsoluble collagen irradiated in nitrogen; O - - O , acid-soluble collagen irradiated in air; i - - ~ , proctase-treated acid-soluble collagen irradiated in nitrogen; k - - & , proctase-treated acidsoluble collagen irradiated in air. The dotted line indicates the range at which collagen solutions gel. Collagen concn. ,o. lO9 % ; temp., 2o ° ; solvent, o.o 5 % acetic acid.
b
O 40O
54
21
2or
lO0
I 25
r 30
,
! 35
T 40
,
i
I 25 45° Temperature
J
I
'
30
35
40
I
i
45°
Fig. 3, Melting curves of ultraviolet irradiated collagen. (a) Irradiated in air: Curve i, native acid-soluble collagen; Curve 2, proctase-treated, 2o rain irradiation; Curve 3, acid-soluble collagen, i h irradiation; Curve 4, proctase-treated, 3 h irradiation; Curve 5, proctase-treated, 5 h irradiation, Curve 6, acid-soluble collagen, 6 h irradiation; Curve 7, acid-soluble collagen, io h irradiation; Curve 8, acid-soluble collagen, 24 h irradiation. (b) Irradiated in nitrogen: Curve 1, native acid-soluble collagen; Curve 1, native acid-soluble collagen; Curve 2, proctasetreated, 2o rain irradiation; Curve 3, acid-soluble collagen, i h irradiation; Curve 4, proctasetreated, 3 h irradiation; Curve 5, proctase-treated, 9 h irradiation; collagen concn., O.lO9%; solvent, 0.05 % acetic acid.
Biochim. Biopkys. Acta, 229 (1971) 672-68o
T. MIYATAet al.
675
treated material are seen during the first 2 h, but after that, melting temperature decreases more rapidly for material irradiated in air than for that irradiated in nitrogen. Melting temperature also decreases more rapidly for acid-soluble than for proctase-treated collagen. All irradiated collagen solutions have a specific optical rotation ((a)n 2°°) higher than --380 (also see Fig. I) after 5-6 h irradiation, indicating retention of helical structure. Melting temperature of irradiated collagen solutions is several degrees lower than that of native acid-soluble collagen, however, and the transition range from helix to coil becomes less distinct. Helical structUre was completely destroyed after 24 h irradiation in air. Native fibrils were reconstituted from collagen solution by incubation at 37 ° under physiologic conditions. Brief irradiation in air results in a marked loss in the ability of native collagen to form fibrils (Fig. 4)- Reconstitution of fibrils has not been observed after 5° min irradiation in air or after 9 ° min irradiation in nitrogen. Even after only 25 min irradiation in air or in nitrogen, the lag time for fibril formation becomes longer and the final values for the extinction coefficient are only about half of that for nonirradiated material.
Effects of ultraviolet irradiation on chemical properties of collagen Amino acid composition of telopeptides isolated from the dialysate of proctasetreated acid-soluble collagen is shown in Table I. The aromatic amino acids, tyrosine and phenylalanine, are removed from acid-soluble collagen by proctase treatment, as are glutamic acid, aspartic acid, serine, alanine, leucine and arginine. 66 amino acid residues are removed from one molecule of acid-soluble collagen by proctase treatment. Moles of leucine equivalents per lO 5 g protein are plotted as a function of time 10Cm 0.~
--
® 8C 0.6
u04
~ 40
o <
~.
A
A
A
02 -6 2 ( ] - -
0~
i
60
120
180
240
l i m e Immutes}
0,0
,
I 10
,
L 20
,
I 30
,
I
i
40
Irradiation time l h 0 u r s l
Fig. 4. Effect of u l t r a v i o l e t i r r a d i a t i o n on fibril f o r m a t i o n f r o m acid-soluble collagen solution. T e m p . , 37°; final collagen concn., o . o 5 % ; final p H , 7.2; final I, o.155; A - - A , n a t i v e acid-soluble collagen; A - - A , i r r a d i a t e d 25 m i n in n i t r o g e n ; O - - O , i r r a d i a t e d 25 m i n in air; 0 - - 0 , i r r a d i a t e d 5 ° m i n in n i t r o g e n ; O - - O , irradiated 5 ° m i n in air. Fig. 5. N i n h y d r i n reaction of u l t r a v i o l e t i r r a d i a t e d collagen as a f u n c t i o n of i r r a d i a t i o n t i m e . A - - / k , acid-soluble collagen i r r a d i a t e d in air; & - - A , p r o c t a s e - t r e a t e d collagen i r r a d i a t e d in air; O - - O , acid-soluble collagen i r r a d i a t e d in n i t r o g e n ; O - - O , p r o c t a s e - t r e a t e d collagen i r r a d i a t e d in nitrogen.
Biochim. Biophys. Acta, 229 (1971) 672-680
677
ULTRAVIOLET IRRADIATION OF COLLAGEN TABLE I AMINO
ACID
SOLUBLE
COMPOSITION
OF TELOPEPTIDES
FROM
DIALYSATE
OF PROCTASE
A-TREATED
ACID-
COLLAGEN
V a l u e s of residues p e r mole of collagen were c a l c u l a t e d u s i n g 26o ooo for t h e m o l e c u l a r w e i g h t of collagen.
A m~no acid
Hydroxylysine Lysine Histidine Arginine A s p a r t i c acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxyproline Ammonia
moles/zo ~g telopept,des
Residues per mole of collagen
28.I 22.6 29.8 93.9 I 2. i 67 , 8 129.7 8o. 5 136. i 45,9 14. 3 4- 7 12. 3 81.9 71.5 57,o -149. 3
o 2 2 2 7 I 5 io 6 io 4 I o I 6 5 4 o o
lO37.5
66
of irradiation in Fig. 5. The values for proctase-treated acid-soluble collagen irradiated in nitrogen show no change with irradiation time, indicating that ninhydrin positive groups are not formed. Acid-soluble collagen irradiated in nitrogen shows a slight increase in color value as irradiation proceeds. Irradiation in air, however, results in many ninhydrin reactive groups. Collagen irradiated for 9, 24, and 58 h was hydrolyzed with 6 M HC1 and amino acids analyzed. Table II shows the amino acid composition of collagen irradiated in air and in nitrogen. Tyrosine and methionine are completely destroyed by 9 h irradiation in both air and nitrogen and phenylalanine is also decomposed after 24 h irradiation. Except for tyrosine, methionine and phenylalanine, the amino acid composition of material irradiated in nitrogen is similar to that of nonirradiated acid-soluble collagen. The recovery of N, however, is decreased to 95 % and amide is increased to 117 moles/io 5 g protein for collagen irradiated for 58 h. Hydroxyproline, proline, lysine, leucine, serine and histidine content are decreased, but that of glutamic acid, aspartic acid and threonine are apparently increased after irradiation in air, as compared to irradiation in nitrogen. Amide content is considerably increased and the recovery of N is decreased after irradiation in air. Thus, destruction of amino acids proceeds more rapidly during irradiation in air than it does during irradiation in nitrogen. A small new peak is observed at the position after phenylalanine in material irradiated for 58 h in air. Biochim. Biophys. Acta, 229 (1971) 672-68o
678
T.
MIYATAet al.
TABLE II AMINO ACID COMPOSITIONSOF ACID -SOLUBLE COLLAGEN AND ULTRAVIOLETIRRADIATED COLLAGENS A m i n o acid
Hydroxylysine Lysine Histidlne Arginine Aspartlc acid Threonlne Serine Glutamic acid Proline Glycine Alanlne Valine Methionine Isoleucme Leucine Tyroslne Phenylalanine Hydroxyproline Amide-N N recovery (%)
Irradmted ~n mtrogen
Irradzated in azr
9h
24 h
58 h
9 h
24 h
58 h
7.2 29.0 4. I 58 .9 46"3 17.1 32.1 68.2 126.8 379.o 121.9 19.1 -12 i 24.0 . 2.9 12o. 4 59.9 98.2
8.5 33.4 4.3 63.5 46"4 18.o 34.8 72.8 133.2 376.8 126. 4 23. 4 -13.o 25.6
7.6 3 °.2 2.9 6o. 2 41.5 18. 4 35.8 75.7 132. 5 373.6 131.8 23.8 -12. 7 25.8 . -12o.2 116.8 94.9
6.7 29.8 4-4 62.2 43 2 18.1 32.0 71.6 129.4 374 .6 13o. 5 2i.i
8. I 3o. 3 3. I 59.3 52. 7 18.8 34.3 78.5 12o.4 379.4 126.1 23. 3
6.9 19.1 -45.3 64.0 22.8 28.9 lO3.5 94.7 379.8 14o.8 23.0
.
. 0.9 121.1 72.8 98.0
Natwe
- -
- -
- -
13.5 25 o
11. 7 27. 9
9.8 15.6
-122.o 138.2 93.5
-91.4 473.6 68.o
.
.
4.5 127.8 78.3 99 .0
5.9 26. 4 3.7 44.9 51.3 18.1 39.2 78.8 127.1 382.6 124.1 23.6 4.3
11.8 26. 3 2.6 13.2 118. 5 60.6 98. 5
Complement fixation studies One of 5 rabbits immunized with proctase-treated collagen developed antibodies specific to collagen, as determined by parallel loss of complement fixing activity and optical rotation with increasing temperature. Fig. 6 illustrates the complement fixation curve for this antibody reacted with collagen, and Fig. 7 illustrates the effects of ultraviolet irradiation. After 9° rain, there is a 9o% loss of complement fixing activity of the collagen. 50 --
50
40
:E
% C' fix
%Chx
20
20
i0
OI
.
I 100
[ 200 Collagen
[ , 300
t 400
.~ 500
(,.t.mg)
0
,
I , I , L L I 20 40 60 80 Duratton of UV irradiation (rain)
,
I 100
Fig. 6. C o m p l e m e n t fixation curve (range of multiple values) of proctase-treated collagen w i t h a 1/200 dilution of a n t i b o d y raised in r a b b i t s to nonirradiated proctase-treated collagen. Fig. 7. Effect of ultraviolet (UV) irradiation on ability of proctase-treated collagen to fix complem e n t w i t h a n t i b o d y raised in r a b b i t s to nonirradiated proctase-treated collagen. B,ochim. Biophys. Acta, 229 (1971) 672-68o
ULTRAVIOLET IRRADIATION OF COLLAGEN
679
DISCUSSION
Tropocollagen molecules contain extrahelicalpe ptide appendages (telopeptides) which are susceptible to proteases, other than collagenase, without any change in the basic molecular structure of collagen9-11. Telopeptides have a markedly different amino acid composition from that of the bulk tropocollagen, most notably in a higher content of tyrosine and phenylalanine. RUBIN et al. 1° and DRAKE et al. n found that pronase is the most effective proteolytic enzyme for digestion of telopeptides and removes 128 amino acids per mole of tropocollagen. Proctase A removes 66 amino acids per mole of tropocollagen. It was calculated that 5 of 7 tyrosine residues were removed by proctase. Proctase A is also an effective enzyme for solubilization of mature insoluble collagen since it is active at a low pH. Viscosity measurements indicate that irradiation in nitrogen induces polymerization of collagen molecules. Melting curves of collagen irradiated for several hours show a broader temperature interval for transition than that of native collagen, in spite of the persistance of a helical content similar to native collagen ((a)D2°° remains close to --400°). Theoretical considerations for the helix-coil transition indicate that increased sharpness of the transition is obtained with increasing molecular weight ~2. The results obtained here thus suggest that scission of collagen molecules into shorter lengths with no destruction of helical configuration has occurred. COOPER AND DAVIDSON1,~have also reported that a fragment of relatively low axial ratio, having a helical configuration, is produced by ultraviolet irradiation of collagen. Irradiation in air induces rapid depolymerization rather than polymerization. Free radicals produced by ultraviolet light probably react with oxygen before participating in cross-linking reactions. Tropocollagen telopeptides play an important role in native fibril formation. Enzyme treated tropocollagen does not readily form fibrils. RUBIN et al. 6 also showed that end-to-end polymerization of tropocollagen was inhibited by pepsin treatment. Ultraviolet light further inhibits fibril formation, suggesting that remaining telopeptides are probably rapidly modified by ultraviolet irradiation. The decrease in complement fixing activity of collagen after ultraviolet irradiation again points to modification of telopeptide regions, since these areas have been found to be important determinants of the immunologic behavior of collagenl3,14. The observations that polymerization of collagen and modifications of telopeptides rich in tyrosine and phenylalanine content occur predominantly during early stages of irradiation suggest that aggregation of collagen molecules is primarily due to combinations between free radicals which result from irradiation of tyrosine and phenylalanine residues. The findings of FORBES AND SULLIVAN1~ that the signals from electron-spin resonance spectra of collagen irradiated at 2,537 A are associated with unpaired electrons located predominantly on the aromatic nuclei of tyrosine and phenylalanine residues support this hypothesis. Amino acid analysis of collagen irradiated for 54 h in air shows decomposition of many amino acids, and higher values of amide, indicating that irradiation in air causes destruction of collagen polypeptide chains. Amide content of irradiated collagen seems to result from amide and ammonia, and the ninhydrin color value results from terminal amino groups and ammonia. Since terminal amino groups would Bzoch~m. B~ophys. Acta, 229 (I97 x) 672-680
680
T. MIYATAet al.
change to amino acids after hydrolysis, the finding that the amide value is always greater than the ninhydrin value (corrected with 60% of color yield of ammonia, based on leucine) suggests that many amides result from a split of the bond between nitrogen and a-carbon H
I
-C-N- -C-
II
O
of the polypeptide chain during irradiation. These results have practical significance in that they are the methods we use for modifying collagen for biomedical applications. Gels formed by ultraviolet irradiation, for instance, have been placed in animal eyes as vitreous implants and have been well tolerated and remain clear TM. The cross-linking effects of ultraviolet irradiation are also used to strengthen collagen membranes used for extra-corporeal dialysis of blood17,18. ACKNOWLEDGEMENTS
This work was supported in part by U.S. Public Health Service grant No. HEo8736, and by Contract No. 69-2044 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service, D.H.E.W.. It was presented, in part, at the Collagen Symposium, Tokyo, Japan,I97O. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 I6 17 I8
D. R. COOPER AND R. J. DAVIDSON, Biochem. J., 97 (1965) 139. D. R. COOPER AND R. J. DAVIDSON, Bioehem. J., 98 (1966) 655. R. J. DAVlDSON AND D. R. COOPER, Bioehem. J., lO 5 (1967) 965. E. FUJIMORI, Biopolymers, 3 (1965) 115. E. FUJIMORI, Biochemistry, 5 (1966) 1934. A. L. I~.UBIN, D. PEAHL, P. T. SPEAKMAN, P. F. DAVlSON AND F. O. SCHMITT, Science, 139 (1963) 37. H. ROSEN, Arch. Biochem. Biophys., 67 (1957) IO. E. WASSERMAN AND L. LEVINE, J. Immunol., 87 (1961) 29o. T. NISHIHARA AND T. MIYATA, Collagen Syrup., 3 (1962) 66. A. L. RUBIN, M. P. DRAKE, P. F. DAVlSON, D. PFAHL, P. T. SPEAKMAN AND F. O. SCHMITT, B,oehemistry, 4 (1965) 181. M. P. DRAKE, P. F. DAVISON, S. BUMP AND F. O. SCHMITT, Biochem,stry, 5 (1966) 3Ol. L. MANDELKERN, Crystallization of Polymers, McGraw-Hill, N e w York, 1964, p. 52. F. O. SCHMITT, L. LEVINE, M. P. DRAKE, A. L. RUBIN, D. PFAHL AND P. F. DAVISON, Proc. Natl. Acad. Sci., U.S., 51 (1964) 493. P. F. DAVISON, L. LEVINE, M. P. DRAKE, A. L. ROBIN AND S. BUMP, J. Exptl. Med., lO 3 (1967) 331 • W. F. FORBES AND P. n . SULLIVAN, Biochim. Biophys. Aeta, 12o (1966) 222. K. H. STENZEL, M. W. DUNN, A. L. RUBIN AND T. MIYATA, Science, 164 (1969) 1281. A. L. RUBIN, R. R. RIGGIO, R. L. 7NACHMAN, G. H. SCHWARTZ, T. MIYATA AND K. H. STENZEL, Trans. Am. Soc. Artificial Internal Organs, 14 (1968) 169. K. H. STENZEL, J. F. SULLIVAN, T. MIYATA AND A. L. ROBIN, Trans. Am. Soc. Artificial Internal Organs, 15 (1969) I14.
Biochzm. Bzophys. Acta. 229 (1971) 672-680
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35807 EFFECTS OF U L T R A V I O L E T IRRADIATION ON NATIVE AND T E L O P E P T I D E - P O O R COLLAGEN
TERUO MIYATA, T A K E S H I SOHDE, ALBERT L. R U B I N AND K U R T H. STENZEL
Japan Leather Co., Ltd., Tokyo (Japan), and the Rogosin Laboratories, Departments of Biochemistry and Surgery, The New York Hospital-Cornell Medical Center, New York, N.Y. (U.S.A.) (Received October 23rd, 197 o)
SUMMARY
Effects of ultraviolet light on physical, chemical and immunologic properties of acid-soluble and enzyme (proctase)-treated collagen were studied. Ultraviolet irradiation in air caused an initial increase in viscosity of both collagen preparations, rapidly followed by a loss of viscosity and a loss of negative optical rotation. Ultraviolet irradiation in nitrogen, however, led to a rapid increase in viscosity until a gel formed. With prolonged irradiation, this gel also depolymerized. Enzyme-treated collagen was less sensitive to the effects of ultraviolet irradiation than was acidsoluble collagen. Immunologic reactivity of enzyme-treated collagen was rapidly lost with short exposures to ultraviolet irradiation. Ultraviolet irradiation also inhibited fiber formation. These results suggest that photochemical modifications occur at telopeptide regions or remaining aromatic residues of enzyme-treated collagen during early stages of irradiation. The transition temperature of the melting curve of collagen became broad but there was no loss of negative optical rotation, suggesting that scission of collagen molecules into shorter fragments which retain helical configurations occurred with longer periods of irradiation. Splitting of the polypeptide chain probably occurs between nitrogen and the a-carbon.
INTRODUCTION
We have attempted to use animal collagen for a variety of biomedical applications. Ultraviolet irradiation appears to be an ideal means of cross-linking and restructuring enzyme solubilized collagen, since it introduces no possibly toxic substituents, and is simple and effective. Several reports indicate that the structure of collagen is modified by ultraviolet irradiation. One effect of ultraviolet irradiation is reported to be a scission of primary collagen polypeptide chains l-a, while other effects appear to be photopolymerization of collagen and fibril formation 4,5. The primary chemical phenomena occurring during the initial stages of these modifications of collagen are thought to Biochim. Biophys. Acta, 229 (1971) 672-680
ULTRAVIOLET IRRADIATION OF COLLAGEN
673
be photochemical reactions involving tyrosine and phenylalanine. The telopeptide or end-chain regions of collagen are relatively rich in aromatic residues e, and also contain important antigenic determinants of collagen. We have determined the effects of ultraviolet irradiation on chemical, physical and immunologic properties of native (acid-soluble) and proctase-treated (telopeptide poor) collagen in both air and nitrogen atmospheres.
MATERIALS AND METHODS
Preparation of acid-soluble collagen Acid-soluble collagen was extracted from calf skin with o.15 M citrate buffer (pH 3.6) and purified by reprecipitation by dialysis against 0.02 M Na2HP04. The reconstituted fiber was then washed with water, dissolved in 0.05% acetic acid and dialyzed against 0.05 % acetic acid.
Proctase treatment of acid-soluble collagen Acid-soluble collagen in 0.05% acetic acid was treated with proctase A* (I/2OO) at 20 ° for 24 h, then dialyzed first against 0.02 M Na,HPO 4 and then against a borax buffer solution (0.037 M Na2C03-o.oi2 M Na,B407, pH IO.O) at 20 ° to inactivate the enzyme. The reconstituted fiber was then washed with water.
Ultraviolet irradiation Collagen solutions (O.lO9% in 0.05% acetic acid) were irradiated at 4 ° with a 4-W ultraviolet lamp (Sankyo Electric Co.) irradiating primarily at 2,537 A. Solutions were placed in IOO ml fused quartz flasks at a distance of io cm from the lamp. Irradiation was carried out either in the presence of air or in oxygen-free nitrogen gas. Nitrogen was passed through the collagen solutions for 30 min before, and then continued during the entire period of irradiation.
Optical rotation Optical rotation was measured at 20 4- o.i ° in a 2o-cm water-iacketed tube in a Rudolf (Model 8o) photoelectric polarimeter equipped with an oscillating polarizer pi'ism. Readings were taken at 589 m/~. Melting curves were determined by measuring optical rotation of collagen solutions as a function of temperature. The temperature of the solutions was raised at a rate of I°/IO min.
Viscosity Viscosities were measured at 20 4 - o . o i ° in an Ostwald-Fenske viscometer having a water flow time of 200 sec at 20 ° and a velocity gradient of I,OOO sec -1.
* Proctase A is an acidic proteolytic enzyme obtained from the incubation b r o t h of A spergillus niger var. macrosporous, has a p H o p t i m u m of 3.0 and was kindly supplied b y Dr. Koaze of Meiji Seika Kaisha, Ltd.
Biochim. Biophys. Acta, 229 (1971) 672-680
674
T. MIYATAet al.
Amino acid analysis Amino acid compositions were determined in a Hitachi amino acid analyzer Model KLA-2 following hydrolysis with 6 M HC1 under vacuum at i i o ° for 16 h.
Ninhydrin reactions Colorimetric determinations of irradiated collagen solutions with ninhydrin were done using a leucine standard.
Fibril formation Collagen solutions (0.1%) in 0.05% acetic acid were preheated at 3 °° for 2 h and mixed with an equal volume of phosphate buffer (0.o4 M Na~HPO4-o. 3 M KH2PO4-o.27 M NaC1, pH 8.5) preheated to 37 °. After mixing, extinction of collagen solutions at 4o0 m# was measured as a function of time at'37 ° in a Beckman DUtype spectrophotometer. The final mixture had a pH of 7.2, an ionic strength of o.155 and a collagen concentration of 0.o50/0 .
Complement fixation Complement fixation studies were done by the method of WASSERMAN AND LEVINE8 utilizing antibodies produced in rabbits to proctase-treated acid-soluble collagen.
RESULTS
Effects of ultraviolet irradiation on physical properties of collagen Optical rotation of acid-soluble and proctase-treated acid-soluble collagen was measured as a function of time of ultraviolet irradiation in both air and nitrogen atmospheres and results are shown in Fig. I. The specific rotation of acid-soluble collagen decreased more rapidly than that of proctase-treated acid-soluble collagen when irradiated either in air or in nitrogen. Irradiation in air caused more rapid degradation of the collagen triple helix, as indicated by the sharp decrease in specific rotation after 5 h irradiation for acid-soluble collagen and after IO h irradiation for proctase-treated acid-soluble collagen. Reduced viscosity of acid-soluble collagen and proctase-treated acid-soluble collagen irradiated in both air and nitrogen are depicted in Fig. 2. Irradiation in nitrogen caused a rapid increase in viscosity and gels were formed after 2 h irradiation of acid-soluble collagen and after 4 h irradiation of proctase-treated acid-soluble collagen. Acid-soluble collagen gels melted after 17 h irradiation and proctase-treated acid-soluble collagen gels melted after IO h irradiation. Thus, during early stages of irradiation, cross-linking reactions between collagen molecules appear to predominate, whereas with prolonged irradiation, degradation occurs. Irradiation in air, rather than in nitrogen, results in a slower increase in viscosity of both collagen preparations, but gels do not form and viscosity begins to decrease after 4 h irradiation. Thus, depolymerization, rather than cross-linking, seems to predominate when irradiation is done in air. Melting temperature decreases as irradiation time increases, as illustrated in Fig. 3. No significant differences between melting curves of untreated and proctaseBiochim. Biophys. Acta, 229 (I97 I) 672-68o
675
ULTRAVIOLET IRRADIATION OF COLLAGEN
40@ 60
'
20C
I00
0
I0
0
20
30
40
50
lrradlatton tlme ihours)
l0
ZO 30 Irradlatlon tlme thours)
40
Fig. I. Effect of ultraviolet irradiation on optical rotation of collagen solutions. 0 - - 0 , acidsoluble collagen irradiated in air; 0 - - - 0 , acid-soluble collagen irradiated in nitrogen; A - - A , proctase-treated acid-soluble collagen irradiated in air; A - - A , proctase-treated acid-soluble collagen irradiated in nitrogen. Collagen concn., o.Io9%; temp., 2o°; solvent, o.o 5 % acetic acid. Fig. 2. Effect of ultraviolet irradiation on reduced viscosity of collagen solutions. O - - O , acidsoluble collagen irradiated in nitrogen; O - - O , acid-soluble collagen irradiated in air; i - - ~ , proctase-treated acid-soluble collagen irradiated in nitrogen; k - - & , proctase-treated acidsoluble collagen irradiated in air. The dotted line indicates the range at which collagen solutions gel. Collagen concn. ,o. lO9 % ; temp., 2o ° ; solvent, o.o 5 % acetic acid.
b
O 40O
54
21
2or
lO0
I 25
r 30
,
! 35
T 40
,
i
I 25 45° Temperature
J
I
'
30
35
40
I
i
45°
Fig. 3, Melting curves of ultraviolet irradiated collagen. (a) Irradiated in air: Curve i, native acid-soluble collagen; Curve 2, proctase-treated, 2o rain irradiation; Curve 3, acid-soluble collagen, i h irradiation; Curve 4, proctase-treated, 3 h irradiation; Curve 5, proctase-treated, 5 h irradiation, Curve 6, acid-soluble collagen, 6 h irradiation; Curve 7, acid-soluble collagen, io h irradiation; Curve 8, acid-soluble collagen, 24 h irradiation. (b) Irradiated in nitrogen: Curve 1, native acid-soluble collagen; Curve 1, native acid-soluble collagen; Curve 2, proctasetreated, 2o rain irradiation; Curve 3, acid-soluble collagen, i h irradiation; Curve 4, proctasetreated, 3 h irradiation; Curve 5, proctase-treated, 9 h irradiation; collagen concn., O.lO9%; solvent, 0.05 % acetic acid.
Biochim. Biopkys. Acta, 229 (1971) 672-68o
T. MIYATAet al.
675
treated material are seen during the first 2 h, but after that, melting temperature decreases more rapidly for material irradiated in air than for that irradiated in nitrogen. Melting temperature also decreases more rapidly for acid-soluble than for proctase-treated collagen. All irradiated collagen solutions have a specific optical rotation ((a)n 2°°) higher than --380 (also see Fig. I) after 5-6 h irradiation, indicating retention of helical structure. Melting temperature of irradiated collagen solutions is several degrees lower than that of native acid-soluble collagen, however, and the transition range from helix to coil becomes less distinct. Helical structUre was completely destroyed after 24 h irradiation in air. Native fibrils were reconstituted from collagen solution by incubation at 37 ° under physiologic conditions. Brief irradiation in air results in a marked loss in the ability of native collagen to form fibrils (Fig. 4)- Reconstitution of fibrils has not been observed after 5° min irradiation in air or after 9 ° min irradiation in nitrogen. Even after only 25 min irradiation in air or in nitrogen, the lag time for fibril formation becomes longer and the final values for the extinction coefficient are only about half of that for nonirradiated material.
Effects of ultraviolet irradiation on chemical properties of collagen Amino acid composition of telopeptides isolated from the dialysate of proctasetreated acid-soluble collagen is shown in Table I. The aromatic amino acids, tyrosine and phenylalanine, are removed from acid-soluble collagen by proctase treatment, as are glutamic acid, aspartic acid, serine, alanine, leucine and arginine. 66 amino acid residues are removed from one molecule of acid-soluble collagen by proctase treatment. Moles of leucine equivalents per lO 5 g protein are plotted as a function of time 10Cm 0.~
--
® 8C 0.6
u04
~ 40
o <
~.
A
A
A
02 -6 2 ( ] - -
0~
i
60
120
180
240
l i m e Immutes}
0,0
,
I 10
,
L 20
,
I 30
,
I
i
40
Irradiation time l h 0 u r s l
Fig. 4. Effect of u l t r a v i o l e t i r r a d i a t i o n on fibril f o r m a t i o n f r o m acid-soluble collagen solution. T e m p . , 37°; final collagen concn., o . o 5 % ; final p H , 7.2; final I, o.155; A - - A , n a t i v e acid-soluble collagen; A - - A , i r r a d i a t e d 25 m i n in n i t r o g e n ; O - - O , i r r a d i a t e d 25 m i n in air; 0 - - 0 , i r r a d i a t e d 5 ° m i n in n i t r o g e n ; O - - O , irradiated 5 ° m i n in air. Fig. 5. N i n h y d r i n reaction of u l t r a v i o l e t i r r a d i a t e d collagen as a f u n c t i o n of i r r a d i a t i o n t i m e . A - - / k , acid-soluble collagen i r r a d i a t e d in air; & - - A , p r o c t a s e - t r e a t e d collagen i r r a d i a t e d in air; O - - O , acid-soluble collagen i r r a d i a t e d in n i t r o g e n ; O - - O , p r o c t a s e - t r e a t e d collagen i r r a d i a t e d in nitrogen.
Biochim. Biophys. Acta, 229 (1971) 672-680
677
ULTRAVIOLET IRRADIATION OF COLLAGEN TABLE I AMINO
ACID
SOLUBLE
COMPOSITION
OF TELOPEPTIDES
FROM
DIALYSATE
OF PROCTASE
A-TREATED
ACID-
COLLAGEN
V a l u e s of residues p e r mole of collagen were c a l c u l a t e d u s i n g 26o ooo for t h e m o l e c u l a r w e i g h t of collagen.
A m~no acid
Hydroxylysine Lysine Histidine Arginine A s p a r t i c acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxyproline Ammonia
moles/zo ~g telopept,des
Residues per mole of collagen
28.I 22.6 29.8 93.9 I 2. i 67 , 8 129.7 8o. 5 136. i 45,9 14. 3 4- 7 12. 3 81.9 71.5 57,o -149. 3
o 2 2 2 7 I 5 io 6 io 4 I o I 6 5 4 o o
lO37.5
66
of irradiation in Fig. 5. The values for proctase-treated acid-soluble collagen irradiated in nitrogen show no change with irradiation time, indicating that ninhydrin positive groups are not formed. Acid-soluble collagen irradiated in nitrogen shows a slight increase in color value as irradiation proceeds. Irradiation in air, however, results in many ninhydrin reactive groups. Collagen irradiated for 9, 24, and 58 h was hydrolyzed with 6 M HC1 and amino acids analyzed. Table II shows the amino acid composition of collagen irradiated in air and in nitrogen. Tyrosine and methionine are completely destroyed by 9 h irradiation in both air and nitrogen and phenylalanine is also decomposed after 24 h irradiation. Except for tyrosine, methionine and phenylalanine, the amino acid composition of material irradiated in nitrogen is similar to that of nonirradiated acid-soluble collagen. The recovery of N, however, is decreased to 95 % and amide is increased to 117 moles/io 5 g protein for collagen irradiated for 58 h. Hydroxyproline, proline, lysine, leucine, serine and histidine content are decreased, but that of glutamic acid, aspartic acid and threonine are apparently increased after irradiation in air, as compared to irradiation in nitrogen. Amide content is considerably increased and the recovery of N is decreased after irradiation in air. Thus, destruction of amino acids proceeds more rapidly during irradiation in air than it does during irradiation in nitrogen. A small new peak is observed at the position after phenylalanine in material irradiated for 58 h in air. Biochim. Biophys. Acta, 229 (1971) 672-68o
678
T.
MIYATAet al.
TABLE II AMINO ACID COMPOSITIONSOF ACID -SOLUBLE COLLAGEN AND ULTRAVIOLETIRRADIATED COLLAGENS A m i n o acid
Hydroxylysine Lysine Histidlne Arginine Aspartlc acid Threonlne Serine Glutamic acid Proline Glycine Alanlne Valine Methionine Isoleucme Leucine Tyroslne Phenylalanine Hydroxyproline Amide-N N recovery (%)
Irradmted ~n mtrogen
Irradzated in azr
9h
24 h
58 h
9 h
24 h
58 h
7.2 29.0 4. I 58 .9 46"3 17.1 32.1 68.2 126.8 379.o 121.9 19.1 -12 i 24.0 . 2.9 12o. 4 59.9 98.2
8.5 33.4 4.3 63.5 46"4 18.o 34.8 72.8 133.2 376.8 126. 4 23. 4 -13.o 25.6
7.6 3 °.2 2.9 6o. 2 41.5 18. 4 35.8 75.7 132. 5 373.6 131.8 23.8 -12. 7 25.8 . -12o.2 116.8 94.9
6.7 29.8 4-4 62.2 43 2 18.1 32.0 71.6 129.4 374 .6 13o. 5 2i.i
8. I 3o. 3 3. I 59.3 52. 7 18.8 34.3 78.5 12o.4 379.4 126.1 23. 3
6.9 19.1 -45.3 64.0 22.8 28.9 lO3.5 94.7 379.8 14o.8 23.0
.
. 0.9 121.1 72.8 98.0
Natwe
- -
- -
- -
13.5 25 o
11. 7 27. 9
9.8 15.6
-122.o 138.2 93.5
-91.4 473.6 68.o
.
.
4.5 127.8 78.3 99 .0
5.9 26. 4 3.7 44.9 51.3 18.1 39.2 78.8 127.1 382.6 124.1 23.6 4.3
11.8 26. 3 2.6 13.2 118. 5 60.6 98. 5
Complement fixation studies One of 5 rabbits immunized with proctase-treated collagen developed antibodies specific to collagen, as determined by parallel loss of complement fixing activity and optical rotation with increasing temperature. Fig. 6 illustrates the complement fixation curve for this antibody reacted with collagen, and Fig. 7 illustrates the effects of ultraviolet irradiation. After 9° rain, there is a 9o% loss of complement fixing activity of the collagen. 50 --
50
40
:E
% C' fix
%Chx
20
20
i0
OI
.
I 100
[ 200 Collagen
[ , 300
t 400
.~ 500
(,.t.mg)
0
,
I , I , L L I 20 40 60 80 Duratton of UV irradiation (rain)
,
I 100
Fig. 6. C o m p l e m e n t fixation curve (range of multiple values) of proctase-treated collagen w i t h a 1/200 dilution of a n t i b o d y raised in r a b b i t s to nonirradiated proctase-treated collagen. Fig. 7. Effect of ultraviolet (UV) irradiation on ability of proctase-treated collagen to fix complem e n t w i t h a n t i b o d y raised in r a b b i t s to nonirradiated proctase-treated collagen. B,ochim. Biophys. Acta, 229 (1971) 672-68o
ULTRAVIOLET IRRADIATION OF COLLAGEN
679
DISCUSSION
Tropocollagen molecules contain extrahelicalpe ptide appendages (telopeptides) which are susceptible to proteases, other than collagenase, without any change in the basic molecular structure of collagen9-11. Telopeptides have a markedly different amino acid composition from that of the bulk tropocollagen, most notably in a higher content of tyrosine and phenylalanine. RUBIN et al. 1° and DRAKE et al. n found that pronase is the most effective proteolytic enzyme for digestion of telopeptides and removes 128 amino acids per mole of tropocollagen. Proctase A removes 66 amino acids per mole of tropocollagen. It was calculated that 5 of 7 tyrosine residues were removed by proctase. Proctase A is also an effective enzyme for solubilization of mature insoluble collagen since it is active at a low pH. Viscosity measurements indicate that irradiation in nitrogen induces polymerization of collagen molecules. Melting curves of collagen irradiated for several hours show a broader temperature interval for transition than that of native collagen, in spite of the persistance of a helical content similar to native collagen ((a)D2°° remains close to --400°). Theoretical considerations for the helix-coil transition indicate that increased sharpness of the transition is obtained with increasing molecular weight ~2. The results obtained here thus suggest that scission of collagen molecules into shorter lengths with no destruction of helical configuration has occurred. COOPER AND DAVIDSON1,~have also reported that a fragment of relatively low axial ratio, having a helical configuration, is produced by ultraviolet irradiation of collagen. Irradiation in air induces rapid depolymerization rather than polymerization. Free radicals produced by ultraviolet light probably react with oxygen before participating in cross-linking reactions. Tropocollagen telopeptides play an important role in native fibril formation. Enzyme treated tropocollagen does not readily form fibrils. RUBIN et al. 6 also showed that end-to-end polymerization of tropocollagen was inhibited by pepsin treatment. Ultraviolet light further inhibits fibril formation, suggesting that remaining telopeptides are probably rapidly modified by ultraviolet irradiation. The decrease in complement fixing activity of collagen after ultraviolet irradiation again points to modification of telopeptide regions, since these areas have been found to be important determinants of the immunologic behavior of collagenl3,14. The observations that polymerization of collagen and modifications of telopeptides rich in tyrosine and phenylalanine content occur predominantly during early stages of irradiation suggest that aggregation of collagen molecules is primarily due to combinations between free radicals which result from irradiation of tyrosine and phenylalanine residues. The findings of FORBES AND SULLIVAN1~ that the signals from electron-spin resonance spectra of collagen irradiated at 2,537 A are associated with unpaired electrons located predominantly on the aromatic nuclei of tyrosine and phenylalanine residues support this hypothesis. Amino acid analysis of collagen irradiated for 54 h in air shows decomposition of many amino acids, and higher values of amide, indicating that irradiation in air causes destruction of collagen polypeptide chains. Amide content of irradiated collagen seems to result from amide and ammonia, and the ninhydrin color value results from terminal amino groups and ammonia. Since terminal amino groups would Bzoch~m. B~ophys. Acta, 229 (I97 x) 672-680
680
T. MIYATAet al.
change to amino acids after hydrolysis, the finding that the amide value is always greater than the ninhydrin value (corrected with 60% of color yield of ammonia, based on leucine) suggests that many amides result from a split of the bond between nitrogen and a-carbon H
I
-C-N- -C-
II
O
of the polypeptide chain during irradiation. These results have practical significance in that they are the methods we use for modifying collagen for biomedical applications. Gels formed by ultraviolet irradiation, for instance, have been placed in animal eyes as vitreous implants and have been well tolerated and remain clear TM. The cross-linking effects of ultraviolet irradiation are also used to strengthen collagen membranes used for extra-corporeal dialysis of blood17,18. ACKNOWLEDGEMENTS
This work was supported in part by U.S. Public Health Service grant No. HEo8736, and by Contract No. 69-2044 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service, D.H.E.W.. It was presented, in part, at the Collagen Symposium, Tokyo, Japan,I97O. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 I6 17 I8
D. R. COOPER AND R. J. DAVIDSON, Biochem. J., 97 (1965) 139. D. R. COOPER AND R. J. DAVIDSON, Bioehem. J., 98 (1966) 655. R. J. DAVlDSON AND D. R. COOPER, Bioehem. J., lO 5 (1967) 965. E. FUJIMORI, Biopolymers, 3 (1965) 115. E. FUJIMORI, Biochemistry, 5 (1966) 1934. A. L. I~.UBIN, D. PEAHL, P. T. SPEAKMAN, P. F. DAVlSON AND F. O. SCHMITT, Science, 139 (1963) 37. H. ROSEN, Arch. Biochem. Biophys., 67 (1957) IO. E. WASSERMAN AND L. LEVINE, J. Immunol., 87 (1961) 29o. T. NISHIHARA AND T. MIYATA, Collagen Syrup., 3 (1962) 66. A. L. RUBIN, M. P. DRAKE, P. F. DAVlSON, D. PFAHL, P. T. SPEAKMAN AND F. O. SCHMITT, B,oehemistry, 4 (1965) 181. M. P. DRAKE, P. F. DAVISON, S. BUMP AND F. O. SCHMITT, Biochem,stry, 5 (1966) 3Ol. L. MANDELKERN, Crystallization of Polymers, McGraw-Hill, N e w York, 1964, p. 52. F. O. SCHMITT, L. LEVINE, M. P. DRAKE, A. L. RUBIN, D. PFAHL AND P. F. DAVISON, Proc. Natl. Acad. Sci., U.S., 51 (1964) 493. P. F. DAVISON, L. LEVINE, M. P. DRAKE, A. L. ROBIN AND S. BUMP, J. Exptl. Med., lO 3 (1967) 331 • W. F. FORBES AND P. n . SULLIVAN, Biochim. Biophys. Aeta, 12o (1966) 222. K. H. STENZEL, M. W. DUNN, A. L. RUBIN AND T. MIYATA, Science, 164 (1969) 1281. A. L. RUBIN, R. R. RIGGIO, R. L. 7NACHMAN, G. H. SCHWARTZ, T. MIYATA AND K. H. STENZEL, Trans. Am. Soc. Artificial Internal Organs, 14 (1968) 169. K. H. STENZEL, J. F. SULLIVAN, T. MIYATA AND A. L. ROBIN, Trans. Am. Soc. Artificial Internal Organs, 15 (1969) I14.
Biochzm. Bzophys. Acta. 229 (1971) 672-680