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The presence of chondroitin sulphate in the synovial fluid in rheumatoid arthritis
CLINICA CHIMICA ACTA
THE
PRESENCE
FLUID
II.5
OF CHONDROITIN
IN RHEUMATOID
P. SEPP:kL& Department (Received
A. LEHTONEN,
sf Medical August
joth,
SULPHATE
IN THE
SYNOVIAL
ARTHRITIS
J. K~RICItAlNEN
Chemistry,
University
of Turku
AND
V.
N.&NTii
(Finlandl
1966)
SUMMARY
The
glycosaminoglycans
were isolated
from
rheumatoid
synovial
fluid and
divided in two fractions by stepwise resolution of their cetylpyridinium complexes on a cellulose column and by electrophoresis. Analvses revealed that the fractions contained in addition to hyaluronic acid, chondroitin 4-sulphate in an amount representing
about 4% of the weight of the total glycosaminoglycans.
The principal
glycosaminoglycan
of human
synovial
fluid is hyaluronic
acid
which was first isolated in 1939 by Meyer et a1.l. The presence of other glycosamino glycans in human synovial fluid has been suggested by other authors2-4. Nanto et al. isolated by stepwise resolution of the cetylpyridinium complexes of the glycosamino glycans in rheumatoid synovial fluid a small fraction that was eluted by a higher salt concentration than hyaluronic acid. Sandson et aL4 prepared an antiserum against the heavy protein polysaccharide (PP-H) of normal human knee articular cartilage. With this antiserum they detected the presence of a compound immunologically very similar to PP-H in normal and in inflammatory synovial fluid. PP-H is a chrondromucoprotein which contains chondroitin sulphate. The presence of chondroitin sulphate in the synovial fluid was not confirmed by chemical methods. This paper describes the isolation of chondroitin sulphate from synovial fluids of patients suffering from rheumatoid arthritis. Some of the results of the study have been presented in a preliminary communication 5. MATERIAL
AND METHODS
Samples The synovial fluids were obtained from three patients suffering from classical or definite rheumatoid arthritis, by puncture of the knee joints. The fluids were pooled to give a final volume of 18 ml.
Isolation The pooled synovial
fluid was hydrolyzed
with papain
(Papain
Clin. Chim.
Crude Powder,
Acta,
16 (I) rrg-119
Sigma Chemical Co., St. Louis, Missouri, U.S.A.) as described by Schilier and cull.6 The amount of papain added to the synovial fluid was 180 mg. The reaction mixture (pH 5.5) was 0.1 M in acetate buffer, o.005 iVl in cysteine and 0.005 ibr in disodium versenate. The incubation time was 48 h and the temperature 60”. The hydrolysate was deproteinized by adding trichloroacetic acid to a final concentration of IO:/: (w/v). After centrifugation, the trichloroacetic acid was removed by shaking the mixture with several portions of ether in a funnel. Finally the glycosaminoglycaxls were precipitated at 4” with 4 volumes of 960/ ethanol which contained 0.50/b sodium acetate and the precipitate was dissolved in 6 ml of distilled water.
of the cetylpyridinium complexes of isolated glycosaminoglycans (cetylpyridinium chloride, Recip Ab, Stockholm, Sweden) was performed in a cellulose column according to procedure of Scott?. The elution was carried out with 0.25 Z and 1.5 N magnesium chloride solutions.
of the isolated glycosaminoglycans was performed on Oxoid@ cellulose acetate sheets (Courtaulds Ltd., Coventry, England), using a barbiturate buffer pH 8.6, and a voltage gradient of IO V/cm during a period of 25 min.* The sheets were then stained with AIcian Blue (I:;‘, (w/v) solution) in 25% (w/v) acetic acid (Gurr Ltd., London G.B.). Hyaluronic. acid, isolated from human umbilical cord, and chondroitin sul#ate, isolated from guinea pig skin, were used as reference compounds in the electrophoresis. Chemical determinations Hexosamine was determined by the Elson-Morgan reaction according to the modi~cation of Boas@ and ~-~~e~yl~~~osa~L~~e by the method of Reissig et al.l”. The ratio of ga~a~~osurn~~~ to g~,~~os~rn~n~was determined by gas chromatography according to a modified method of K~rkk~inen et c$.~~.Uranic acids were analysed by the carbazole method of Bitter and .Muir~2 and by the orcinol. method described by DischeX”. S&..hafe was determined by a modified benzidine methodI and nitrogen by Kjeldahl’s method followed by nesslerization. RESULTS
Fig. I shows the electrophoretic pattern of the unfractionated glycosamino&cans isoIated from the pooled synovial fluid. There are two bands whose efectrophoretic mobilities correspond to those of hyaluronic acid and chondroitin sulphate. The staining of hyaluronic acid was llnnsually weak because of the large amount of glycosaminogIy~alls applied to the celIulose sheet 16. The eIe~trophoretic pattern of the glycosaminoglycans after their fractionation with cetylpyridinium chloride and the pattern of chondroitin sulphate isolated from guinea pig skin are seen in Fig. 2. The results of chemical analyses of the isolated unfractionated glycosaminoglycans are shown in Table I and the results after the CPC-fractionation in Table II. The amount of sulphate in the fraction &ted by 1.5 N l~~a~esiurn chloride was so low that the quantitative determination was unreliable, but there is no doubt about
~HONDR~ITI~
SULPHATE
IN RHEUMATOID
ARTHRITIS
117
the presence of sulphate in this fraction. The molar ratios of hexosamine, hexuronic acid and nitrogen were 1.00:0.98:1.rg. Gas chromatography of the hexosamines of unfractionated glycosaminoglycans is presented in Fig. 3. Galactosamine amounts to 3.3% of total hexosamines. The amount of chondroitin sulphate in the total glycosaminoglycans calculated from the different analytical data is approximately 3.@ (Table III).
Ch-SO,
+-Start
Fig. T. The electrophoretic pattern of unfractionat~d gl~cosamino~l~cans isolated from rheumatoid synovial fluid and stained with Alcian blue. H.4 = hyaluronic acid; Ch-SO., -= chondroitin suiphatc. Fig. 2. Electrophoretic patterns (stained with Alcian blue) of glycosaminoglycans isolated from rheumatoid synovial Auid and guinea pig skin after fractionation with CPC. The norrnalitics of the magnesium chloride solutions used in the clution of the CPC complexes are indrcated. HA = hyaluronic acid ; Ch-SO, = chondroitin sulphate.
CHEMICAL SYNOVIAL
ANALPSIS FLUID
OF UNFRACTIONATED
Hexosamine I-fexuronic acid Carbazolc method Orcinol method Sulphate Nitrogen TABLE
CLYCDSA~*1I~OGLyC.~NS ISOL.XTED FROM
61.0
34 1
64.5 60.0 1.00 5.50
332 309 10 392
RHE~l~i~~TOlD
II
ANALYSIS OF CPC-FRACTIONATED R~EU~l,~TOID SYNOVIAL FLUKD
CHFaICAL
Fraction
Soluble in 0.25 N MgCl, (Hyaluronic acid) Soluble in 1.5 S MgCI, (Chondroitin sulphate)
GLYCO~AWINOGLYCANS
Hexesamine
Hexwonic
pM/mE
pM/ml (carbazole)
2.6
2.3 0.13
0.x.3
ISOLATEI) FRohI
ncid
1.5
ml
OF
Sulphate
-k
--
_-..l-“-C&z. Chim.
Acfa,
16 (I) rrg-x~p
TABLE
Ill
TNE PERCENTAGEOF CHoNDRoITfN SULPWATEIN RHEUMATOID
SYNOVIAL
GLYCOSAMINOGLYCANS
ISOLATED
FROM
FLUID
Calculated from Sulphate content (Table I) Hexosamine content (Table II) Galactosamine content (Fig. 3) -
2.9 4.7 3.3
--
A further analysis of the type of chondroitin sulphate was attempted. No iduronic acid was found by gas chromatographic analysis18 in the fraction soluble in 1.5 N MgCl,. The “acetylhexosamine” in the same fraction determined after digestion with hyaluronidasel’ (Hyaluronidase from bovine testes, Sigma Chemical Co., St. Louis, MO., U.S.A.) amounted to 4.2% of the total hexosamine. According to this the fraction soluble in 1.5 N MgCl, is mainly chondroitin q-sulphate.
5
IO
15
20
25
MINUTES
Pig. 3. Gas chrornato~am of the hexosamines derived from ~lycosa~nino~l~-cans isolated rheumatoid synovial fluid. Stationary phase : I t$, SE-30. Column temperature: 150°.
from
DISCUSSIOK
The glycosaminoglycans are components of the ground substance of connective tissue. The synovial cavity is lined with connective tissue and therefore the presence of glycosaminoglycans other than hyaluronic acid is expected. The sulphate-containing glycosamin~glycan which was isolated from the human rheumatoid synovial fluid was principally chondroitin 4-sulphate. It represented about 4% of the total glycosaminoglycans. The origin of the chondroitin sulphate of synovial fluid is obscure. There are at least three possible sources: (I) articular cartilage, (2) plasma or (3) the synovial membrane where hyaluronic acid is synthetized. In rheumatoid arthritis, where cartilage destruction is usually observed, the first possibility must be taken into consideration. Chondroitin 4-sulphate is known to be present in cartilage and bone, aithough CEi12.Chim. ilcta, 16 (I)
IIj-IIg
CHONDROITIN
SULPHATE
IN RHEUMATOID
Solheim18 has shown that chondroitin cartilage.
In accordance
ARTHRITIS
6-sulphate
119
predominates
in human semilunar
with the studies of Sandson et aL4 we suggest that chondroitin
sulphate found in rheumatoid synovial fluid is released from articular cartilage. On the other hand, chondroitin +sulphate identical with that derived from cartilage has been found in a concentration of about 1.5 mg per liter in normal human plasmaIs. The plasma and synovial fluid chondroitin sulphate may have a common origin. This. is suggested by the studies of Bryant et aLzO, who showed that intravenously injected papain causes the release of chondroitin
sulphate
from rabbit
cartilage
into the blood
stream. ACKNOWLEDGEMENT
This investigation
was supported
by the Reumaliitto
Association), Helsinki, Finland, and by a PHS research National Heart Institute, Bethesda, Md., U.S.A.
(Finnish
Rheumatism
grant HE-06818-03
from the
ADDENDUM
After this paper had been submitted, S. A. Barker, C. F. Hawkins and M. Hewins (Ann. Rheumat. Dis., 25 (1966) 209) have also demonstrated the presence of chondroitin sulphate in rheumatoid synovial fluid. REFERENCES I K. MEYER, E. M. SMYTH AND M. H. DAWSON, J. Biol. Chem., 128 (1939) 319. Rheumat., 3 (1960) Jog. J. E. SMITH, G. T. CROWLEY, JR AND R. B. GILES, JR. , .4rthritis V. NKNTB, P. SEPPXLX AND E. KULONEN, C&n. Chim. Acta, 7 (1962) 794. J. SANDSON, L. ROSENBERG, D. WHITE AND H. SCHUSTER, Arthritis Rheumat., 6 (1963) 791. P. SEPPHLK, Duodecim, 81 (1965) 1049. S. SCHILLER, G. A. SLOVER AND A. DORFMAN, J. Biol. Chem., 236 (1961) 983. 7 J. E. SCOTT, in D. GLICK (Ed.), Methods in Biochemical Analysis. Vol. 8, Interscience, New York, 1960, P. 145. 8 V. N:iNTo, Acta Chem. Stand., 17 (1963) 857. g N. F. BOAS, J. Biol. Chem., 204 (1953) 553. IO J. L. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. BioZ. Chem., 217 (1955) 959. II J. KXRKKKINEN, A. LEHTONEN AND T. NIKKARI, J. Chromatog., 20 (1965) 457. 12 T. BITTER AND H. M. MUIR, Anal. Biochem., 4 (1962) 330. 13 Z. DISCHE, in D. GLICK (Ed.), Methods in Biochemical Analysis, Vol. 2, Interscience, New York,
2 3 4 5 6
1955, P. 3’3. C. A. ANTONOPOULOS, Acta Chem. Stand., 16 (1962) V. Nb;~~ii. unpublished observations, 1965. A. LEHTONEN: J. KKRKKXINEN AND E. -H&ITI, to M. B. MATHEWS AND M. INOUYE, Biochim. Biophys. K. SOLHEIM, J. Oslo City Hosp., 15 (1965) 127.~ . Ig S. SCHILLER, Biochim. Biophys. Acta, 28 (1958) 410. 20 J. II. BRYANT, I. G. LEDER AND D. STETTEN, Arch. 14 15 16 r7 18
1521. be pllblished (1966). Acta, __.. =jq (1961) ,I.509.
Biorhem.
Biophys. CZin. Chim.
76 (1958) 122. Acta,
16 (I) 115-119
THE
PRESENCE
FLUID
II.5
OF CHONDROITIN
IN RHEUMATOID
P. SEPP:kL& Department (Received
A. LEHTONEN,
sf Medical August
joth,
SULPHATE
IN THE
SYNOVIAL
ARTHRITIS
J. K~RICItAlNEN
Chemistry,
University
of Turku
AND
V.
N.&NTii
(Finlandl
1966)
SUMMARY
The
glycosaminoglycans
were isolated
from
rheumatoid
synovial
fluid and
divided in two fractions by stepwise resolution of their cetylpyridinium complexes on a cellulose column and by electrophoresis. Analvses revealed that the fractions contained in addition to hyaluronic acid, chondroitin 4-sulphate in an amount representing
about 4% of the weight of the total glycosaminoglycans.
The principal
glycosaminoglycan
of human
synovial
fluid is hyaluronic
acid
which was first isolated in 1939 by Meyer et a1.l. The presence of other glycosamino glycans in human synovial fluid has been suggested by other authors2-4. Nanto et al. isolated by stepwise resolution of the cetylpyridinium complexes of the glycosamino glycans in rheumatoid synovial fluid a small fraction that was eluted by a higher salt concentration than hyaluronic acid. Sandson et aL4 prepared an antiserum against the heavy protein polysaccharide (PP-H) of normal human knee articular cartilage. With this antiserum they detected the presence of a compound immunologically very similar to PP-H in normal and in inflammatory synovial fluid. PP-H is a chrondromucoprotein which contains chondroitin sulphate. The presence of chondroitin sulphate in the synovial fluid was not confirmed by chemical methods. This paper describes the isolation of chondroitin sulphate from synovial fluids of patients suffering from rheumatoid arthritis. Some of the results of the study have been presented in a preliminary communication 5. MATERIAL
AND METHODS
Samples The synovial fluids were obtained from three patients suffering from classical or definite rheumatoid arthritis, by puncture of the knee joints. The fluids were pooled to give a final volume of 18 ml.
Isolation The pooled synovial
fluid was hydrolyzed
with papain
(Papain
Clin. Chim.
Crude Powder,
Acta,
16 (I) rrg-119
Sigma Chemical Co., St. Louis, Missouri, U.S.A.) as described by Schilier and cull.6 The amount of papain added to the synovial fluid was 180 mg. The reaction mixture (pH 5.5) was 0.1 M in acetate buffer, o.005 iVl in cysteine and 0.005 ibr in disodium versenate. The incubation time was 48 h and the temperature 60”. The hydrolysate was deproteinized by adding trichloroacetic acid to a final concentration of IO:/: (w/v). After centrifugation, the trichloroacetic acid was removed by shaking the mixture with several portions of ether in a funnel. Finally the glycosaminoglycaxls were precipitated at 4” with 4 volumes of 960/ ethanol which contained 0.50/b sodium acetate and the precipitate was dissolved in 6 ml of distilled water.
of the cetylpyridinium complexes of isolated glycosaminoglycans (cetylpyridinium chloride, Recip Ab, Stockholm, Sweden) was performed in a cellulose column according to procedure of Scott?. The elution was carried out with 0.25 Z and 1.5 N magnesium chloride solutions.
of the isolated glycosaminoglycans was performed on Oxoid@ cellulose acetate sheets (Courtaulds Ltd., Coventry, England), using a barbiturate buffer pH 8.6, and a voltage gradient of IO V/cm during a period of 25 min.* The sheets were then stained with AIcian Blue (I:;‘, (w/v) solution) in 25% (w/v) acetic acid (Gurr Ltd., London G.B.). Hyaluronic. acid, isolated from human umbilical cord, and chondroitin sul#ate, isolated from guinea pig skin, were used as reference compounds in the electrophoresis. Chemical determinations Hexosamine was determined by the Elson-Morgan reaction according to the modi~cation of Boas@ and ~-~~e~yl~~~osa~L~~e by the method of Reissig et al.l”. The ratio of ga~a~~osurn~~~ to g~,~~os~rn~n~was determined by gas chromatography according to a modified method of K~rkk~inen et c$.~~.Uranic acids were analysed by the carbazole method of Bitter and .Muir~2 and by the orcinol. method described by DischeX”. S&..hafe was determined by a modified benzidine methodI and nitrogen by Kjeldahl’s method followed by nesslerization. RESULTS
Fig. I shows the electrophoretic pattern of the unfractionated glycosamino&cans isoIated from the pooled synovial fluid. There are two bands whose efectrophoretic mobilities correspond to those of hyaluronic acid and chondroitin sulphate. The staining of hyaluronic acid was llnnsually weak because of the large amount of glycosaminogIy~alls applied to the celIulose sheet 16. The eIe~trophoretic pattern of the glycosaminoglycans after their fractionation with cetylpyridinium chloride and the pattern of chondroitin sulphate isolated from guinea pig skin are seen in Fig. 2. The results of chemical analyses of the isolated unfractionated glycosaminoglycans are shown in Table I and the results after the CPC-fractionation in Table II. The amount of sulphate in the fraction &ted by 1.5 N l~~a~esiurn chloride was so low that the quantitative determination was unreliable, but there is no doubt about
~HONDR~ITI~
SULPHATE
IN RHEUMATOID
ARTHRITIS
117
the presence of sulphate in this fraction. The molar ratios of hexosamine, hexuronic acid and nitrogen were 1.00:0.98:1.rg. Gas chromatography of the hexosamines of unfractionated glycosaminoglycans is presented in Fig. 3. Galactosamine amounts to 3.3% of total hexosamines. The amount of chondroitin sulphate in the total glycosaminoglycans calculated from the different analytical data is approximately 3.@ (Table III).
Ch-SO,
+-Start
Fig. T. The electrophoretic pattern of unfractionat~d gl~cosamino~l~cans isolated from rheumatoid synovial fluid and stained with Alcian blue. H.4 = hyaluronic acid; Ch-SO., -= chondroitin suiphatc. Fig. 2. Electrophoretic patterns (stained with Alcian blue) of glycosaminoglycans isolated from rheumatoid synovial Auid and guinea pig skin after fractionation with CPC. The norrnalitics of the magnesium chloride solutions used in the clution of the CPC complexes are indrcated. HA = hyaluronic acid ; Ch-SO, = chondroitin sulphate.
CHEMICAL SYNOVIAL
ANALPSIS FLUID
OF UNFRACTIONATED
Hexosamine I-fexuronic acid Carbazolc method Orcinol method Sulphate Nitrogen TABLE
CLYCDSA~*1I~OGLyC.~NS ISOL.XTED FROM
61.0
34 1
64.5 60.0 1.00 5.50
332 309 10 392
RHE~l~i~~TOlD
II
ANALYSIS OF CPC-FRACTIONATED R~EU~l,~TOID SYNOVIAL FLUKD
CHFaICAL
Fraction
Soluble in 0.25 N MgCl, (Hyaluronic acid) Soluble in 1.5 S MgCI, (Chondroitin sulphate)
GLYCO~AWINOGLYCANS
Hexesamine
Hexwonic
pM/mE
pM/ml (carbazole)
2.6
2.3 0.13
0.x.3
ISOLATEI) FRohI
ncid
1.5
ml
OF
Sulphate
-k
--
_-..l-“-C&z. Chim.
Acfa,
16 (I) rrg-x~p
TABLE
Ill
TNE PERCENTAGEOF CHoNDRoITfN SULPWATEIN RHEUMATOID
SYNOVIAL
GLYCOSAMINOGLYCANS
ISOLATED
FROM
FLUID
Calculated from Sulphate content (Table I) Hexosamine content (Table II) Galactosamine content (Fig. 3) -
2.9 4.7 3.3
--
A further analysis of the type of chondroitin sulphate was attempted. No iduronic acid was found by gas chromatographic analysis18 in the fraction soluble in 1.5 N MgCl,. The “acetylhexosamine” in the same fraction determined after digestion with hyaluronidasel’ (Hyaluronidase from bovine testes, Sigma Chemical Co., St. Louis, MO., U.S.A.) amounted to 4.2% of the total hexosamine. According to this the fraction soluble in 1.5 N MgCl, is mainly chondroitin q-sulphate.
5
IO
15
20
25
MINUTES
Pig. 3. Gas chrornato~am of the hexosamines derived from ~lycosa~nino~l~-cans isolated rheumatoid synovial fluid. Stationary phase : I t$, SE-30. Column temperature: 150°.
from
DISCUSSIOK
The glycosaminoglycans are components of the ground substance of connective tissue. The synovial cavity is lined with connective tissue and therefore the presence of glycosaminoglycans other than hyaluronic acid is expected. The sulphate-containing glycosamin~glycan which was isolated from the human rheumatoid synovial fluid was principally chondroitin 4-sulphate. It represented about 4% of the total glycosaminoglycans. The origin of the chondroitin sulphate of synovial fluid is obscure. There are at least three possible sources: (I) articular cartilage, (2) plasma or (3) the synovial membrane where hyaluronic acid is synthetized. In rheumatoid arthritis, where cartilage destruction is usually observed, the first possibility must be taken into consideration. Chondroitin 4-sulphate is known to be present in cartilage and bone, aithough CEi12.Chim. ilcta, 16 (I)
IIj-IIg
CHONDROITIN
SULPHATE
IN RHEUMATOID
Solheim18 has shown that chondroitin cartilage.
In accordance
ARTHRITIS
6-sulphate
119
predominates
in human semilunar
with the studies of Sandson et aL4 we suggest that chondroitin
sulphate found in rheumatoid synovial fluid is released from articular cartilage. On the other hand, chondroitin +sulphate identical with that derived from cartilage has been found in a concentration of about 1.5 mg per liter in normal human plasmaIs. The plasma and synovial fluid chondroitin sulphate may have a common origin. This. is suggested by the studies of Bryant et aLzO, who showed that intravenously injected papain causes the release of chondroitin
sulphate
from rabbit
cartilage
into the blood
stream. ACKNOWLEDGEMENT
This investigation
was supported
by the Reumaliitto
Association), Helsinki, Finland, and by a PHS research National Heart Institute, Bethesda, Md., U.S.A.
(Finnish
Rheumatism
grant HE-06818-03
from the
ADDENDUM
After this paper had been submitted, S. A. Barker, C. F. Hawkins and M. Hewins (Ann. Rheumat. Dis., 25 (1966) 209) have also demonstrated the presence of chondroitin sulphate in rheumatoid synovial fluid. REFERENCES I K. MEYER, E. M. SMYTH AND M. H. DAWSON, J. Biol. Chem., 128 (1939) 319. Rheumat., 3 (1960) Jog. J. E. SMITH, G. T. CROWLEY, JR AND R. B. GILES, JR. , .4rthritis V. NKNTB, P. SEPPXLX AND E. KULONEN, C&n. Chim. Acta, 7 (1962) 794. J. SANDSON, L. ROSENBERG, D. WHITE AND H. SCHUSTER, Arthritis Rheumat., 6 (1963) 791. P. SEPPHLK, Duodecim, 81 (1965) 1049. S. SCHILLER, G. A. SLOVER AND A. DORFMAN, J. Biol. Chem., 236 (1961) 983. 7 J. E. SCOTT, in D. GLICK (Ed.), Methods in Biochemical Analysis. Vol. 8, Interscience, New York, 1960, P. 145. 8 V. N:iNTo, Acta Chem. Stand., 17 (1963) 857. g N. F. BOAS, J. Biol. Chem., 204 (1953) 553. IO J. L. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. BioZ. Chem., 217 (1955) 959. II J. KXRKKKINEN, A. LEHTONEN AND T. NIKKARI, J. Chromatog., 20 (1965) 457. 12 T. BITTER AND H. M. MUIR, Anal. Biochem., 4 (1962) 330. 13 Z. DISCHE, in D. GLICK (Ed.), Methods in Biochemical Analysis, Vol. 2, Interscience, New York,
2 3 4 5 6
1955, P. 3’3. C. A. ANTONOPOULOS, Acta Chem. Stand., 16 (1962) V. Nb;~~ii. unpublished observations, 1965. A. LEHTONEN: J. KKRKKXINEN AND E. -H&ITI, to M. B. MATHEWS AND M. INOUYE, Biochim. Biophys. K. SOLHEIM, J. Oslo City Hosp., 15 (1965) 127.~ . Ig S. SCHILLER, Biochim. Biophys. Acta, 28 (1958) 410. 20 J. II. BRYANT, I. G. LEDER AND D. STETTEN, Arch. 14 15 16 r7 18
1521. be pllblished (1966). Acta, __.. =jq (1961) ,I.509.
Biorhem.
Biophys. CZin. Chim.
76 (1958) 122. Acta,
16 (I) 115-119