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Isolation of new polysaccharide sulphates from charonia lampas
16
BIOCHIMICAET BIOPHYSICAACTA
BBA 85034 ISOLATION OF N E W P O L Y S A C C H A R I D E SULPHATES FROM C H A R O N I A LAMPAS
SADAKO INOUE* Department of Biophysics and Biochemistry, Faculty of Scie~zce, University of Tokyo, Tokyo (Japa;z) (Received August 1tth, i964)
SUMMARY A sulphated heteropolysaccharide named horatinsulphuric acid has been isolated from a sulphatase-rich gastropod, Charonia lampas. Horatinsulphuric acid, a mixture of analogous polymers, is obtained by the method of trypaflavin precipitation of sulphate esters. Fractionation made by column chromatography using DEAESephadex separates the mixture into several fractions in which the sulphate content varies from IO to 3o%. The composition of the sugar portion consists of fucose, mannose, glucose, galactose, glucosamine, galactosamine and sialic acid.
INTRODUCTION Charonia lampas, a marine gastropod**, has been noted for its active sulphatasesl,2. The function of the sulphatases in this gastropod has been solved only incompletely3, a. An idea that there must be natural substrates for the sulphatases led to the isolation of a glycan sulphate named charoninsulphuric acid from the mucous gland of this gastropod 5. Recently tissues of C. lampas were re-examined to find any other natural sulphate esters and it was found that the liver contains at least two families of polysaccharide sulphates. One, having the properties of a sulphated heteropolysaccharide, was named horatinsulphuric acid and the other sulphate ester was found to consist of a hitherto unknown sugar. A sulphate ester fraction distinguishable from charoninsulphuric acid was also separated from the mucous gland. This paper describes the isolation and properties of horatinsulphuric acid. A preliminary account of this paper has already appeared 6. EXPERIMENTAL PROCEDURE
The liver of C. lampas was frozen in dry ice-ether immediately after dissection and kept in a deep freezer until needed. - -;-Present address: l)epartment of Biochemistry, Tohoku University, School of Medicine, Sendai (Japan). .... B6shfi-hora" in Japanese. The same species is frequently referred to as Charonia sauliae. 13iochim. Biophys. Acta, ioi (1905) 16 25
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
17
Isolation procedure Step I: Extraction and precipitation by ethanol. The standard procedure was a s follows: The frozen liver (from 12 animals; 600 g) was homogenized with 1.2 1 of water in a Waring blendor. The homogenate was boiled for 5 min and centrifuged at 4000 rev./min for 15 min after cooling. The slimy residue was discarded. To the supernatant was added I volume of ethanol and the mixture was left to precipitate overnight in the cold. The precipitate was collected by centrifugation and dissolved in 15o ml of water. The insoluble material was removed by high-speed centrifugation (IO ooo rev./min for 30 min). The fraction containing horatinsulphuric acid was reprecipitated with 2 volumes of ethanol from the clear solution. The precipitate was washed and dried by the usual method. Yield: IO g. Step 2: Precipitation with trypaflavin. The ethanolic precipitate, IO g, was dissolved in ioo ml of water and 5 ml of a lO% aqueous solution of trypaflavin (acriflavin, 3,6-diamino-Io-ifiethylacridinium chloride) was added. The resulting mixture was left overnight in the cold. The precipitate was collected and washed with water. The washing was repeated (usually 3 times wit h 5o-ml portions of water) until the washing water became almost colourless. The trypaflavin precipitate was dissolved in 300 ml of 5 M NaC1. When 2 volumes of ethanol were added to the solution, horatinsulphuric acid was precipitated with a large amount of NaC1. The precipitate was collected on a sintered glass funnel and washed with ethanol to remove trypaflavin. The washed precipitate was dissolved in 600 ml of water and dialysed against running tap water. The dialysed solution was concentrated on an evaporator to 60 ml at a temperature below 4 °o . Four volumes of ethanol were added to the concentrated solution and the precipitate was collected, washed and dried by the usual method. Yield: 9o0 mg (crude sodium horatinsulphate). The main contaminant of the crude material was nucleic acid, for the latter also gave a precipitate with trypaflavin. The amount of nucleic acid in this preparation was about 30 % when calculated as RNA b y absorbancy at 260 m/,. Step 3: Column chromatography on DEAE-Sephadex. A column (I.O x 20 cm) was prepared with DEAE-Sephadex A-5o, coarse grade, and was washed thoroughly with o.oi M Tris-HC1 buffer (pH 8.0). The crude horatinsulphuric acid (292 rag), dissolved in 5 ml of the buffer was applied to the column and eluted with the buffer. 5-ml fractions were collected at a flow rate of I ml per min. Elutions wer~ successively performed with the buffer containing NaC1 of increasing concentrations. The eluate was analysed for sulphate ester, sialic acid, and ultraviolet-absorbing material b y the method described below. The elution diagram is shown in Fig. I. The fractions in every peak were pooled, concentrated to one-tenth volume b y a rotary evaporator, dialysed and lyophilised. The recovery of ester sulphate was 73 %. Step 4: Re-chromatography by gradient-elution technique. The fractions obtained b y column chromatography on DEAE-Sephadex were further purified b y a second chromatography on a column of DEAE-Sephadex, I.O x 15 cm. A concentration gradient of NaC1 was obtained b y introducing 500 ml of 0.5 M NaC1 into a mixing chamber containing 20o ml of o.oi M Tris-HC1 (pH 8.0). When the 0.5 M NaC1 was used up, 0.75 M NaC1 was added to the reservoir, and 4-ml fractions were collected. Typical elution diagrams obtained from the chromatography of 0.4 M and o.5 M NaC1 eluates from the first column are shown in Fig. 2a and Fig. 2b, respectively. Fractions Biochim. Biophys. Acta, i o i (1965) 16-25
i8
S. I N O U E Fr - I
0.15
Fr-3
Fr-6
0.10
.Q
< o.o~
o
r
50
0
t
Buffer
t
0.1MNaCI
t
0.2MNaCI
100
Fraction number
t
0.3MNaCI
t
OAMNaCI
f
150
0.SMNoCI
20q
t
0.7MNaCI
1MNaCI
Fig. I. F r a c t i o n a t i o n o f h o r a t i n s u l p h u r i c a c i d b y c o l u m n c h r o m a t o g r a p h y o n D E A E - S e p h a d e x . - - - © - - -, s u l p h a t e e s t e r ; D--~, s i a l i c a c i d ; - - © , a b s o r b a n c y a t 260 ml~.
obtained by chromatography on the second column were virtually free from ultraviolet-absorbing material and no trace of ribose was detected on paper chromatograms when the hydrolysates were examined.
Zone electrophoresis on GEON GEON (No. 237 S, Japan GEON Co.) was previously washed with dilute alkali, water and finally with the buffer used for the electrophoresis. The suspension of GEON in the buffer was poured into a plastic trough, 1.5 × 5.0 × 45 cm, placed horizontally. 0.05 M Tris-acetate (pH 6.0) was found to be most satisfactory for the procedure. 0.5 g of the 50% ethanolic precipitate of the liver extract was dissolved in 4.0 ml of the buffer, mixed with dry GEON powder and the resulting paste was poured in a gap of I cm width which was made across the GEON bed at a distance of IO cm from the end of the trough near the cathode. The electrophoresis was carried out for 7 h at 16-18 mA and i . i kV in the cold. After the electrophoresis, the bed of GEON was cut into sections of i cm each in width and each section was eluted with 20 ml of water. Portions of the eluate were examined for the presence of sulphate ester by trypaflavin, ultraviolet-absorbing material, sialic acid and hexose.
Semi-quantitative determination of sulphate ester Although there was a report 7 in which the complex formation between sulphate ester and acridine derivative was used in the estimation of sulphate ester, the method is not commonly used. In the present work, a simple method based on the same idea was effectively used in the detection and semi-quantitative determination of sulphate ester in the eluates from the columns or in the fractions separated by electrophoresis. The procedure is as follows: To 0.5 ml of the solution of the sample containing sulphate ester at a concentration below I/~mole, o. I ml of a 0.5 % aqueous solution of Biochim. Biophys. Acta,
i o i (1965) 1 6 - 2 5
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
°4° F
1o
o.3o~
~ ~t
/
',
Fr-3'
1/\)
o.2ol-
? ~ ~
I
"/ 0
19
20
40
A
,o
÷,~',o\
~
I/?:i... IE:-
60 Fraction number Fr-4'
0.30
F~ 1.0
[cl~ ~' 0.20
~ <
%~
I
,............................. 3.5
0.10
0
0
20
40
60 80 100 120 140 160 180 Fraction number Fig. 2. R e - c h r o m a t o g r a p h y of h o r a t i n s u l p h u r i c acid fractions : a, F r a c t i o n 3 of Fig. i ; b, F r a c t i o n 4 of Fig. i. - - - O - - - , s u l p h a t e ester; - - Q - - , sialic acid; - - ~ - - , hexose b y a n t h r o n e ; - - O - - , a b s o r b a n c y at 260 m/u; . . . . . . . concentration of NaC1.
trypaflavin was added. After 15 min, 2.5 ml of water were added and the turbidity which formed was measured at 660 m/~. As shown in Fig. 3, a linear~relationship between the turbidity and the concentration of sulphate ester was o~tained. The stability of the turbidity was influenced by the concentration of salt in the solution in which the sulphate ester was dissolved. Therefore, when the concentration of salt in the solution of the sample is high, the standard curve must be prepared in the presence of salt at a similar concentration. The measurement shown in Fig. 3 was made in the absence of salt. The method gave satisfactory reproducibility and the variation in the turbidity obtained in solutions of the same concentration was small from one experiment to the other.
Other analytical methods Sulphate was determined by the method of LLOYDs. Neutral sugars were determined by either the phenol-I-I2SO4 method 9 or the anthrone method 1°. A mixture of glucose, galactose and mannose (1:2:1) was used as a standard in the Biochim. Biophys. Acta, IOI (I965) 16-25
2o
s. 1NOUE
0.40
0.30
~0.20
0.10
0
0
I 0.5
mg
1.0
Fig. 3. Determination curve of horatinsulphuric acid by trypaflavin precipitation. The sulphate content of the horatinsulphuric acid preparation is 1.53/,moles SO~- per mg.
phenol-H2SO a reaction; Hexosamine was determined by a modified Elson-Morgan reaction u ; Sialic acid was determined by the thiobarbituric acid reaction according to AMINOFF'S procedurO2; Nitrogen content was estimated with the micro-Kjeldahl apparatus; Reducing power was determined by the method of PARK AND JOHZ~SON~S; Infrared spectra were measured in K B r discs with a Hitachi spectrophotometer.
Electrophoretic and chromatographic techniques Paper electrophoresis and paper chromatography were carried out on W h a t m a n No. 3MM or Toyo 5IA paper. The following buffer and solvent systems were used: Buffer I, o.o5 M Tris-acetate (pH 6.o); Buffer II, o.I M ammonium acetate-acetic acid (pH 4.o); Solvent I, ethylacetate-pyridine-water (2:1:2 v/v), upper phase; Solvent II, n-butanol-pyridine-water (6:4:3 v/v); Solvent I I I , n - b u t a n o l - n p r o p a n o l - o . i N HC1 ( i : 2 : i v / v ) ; Solvent IV, n - b u t a n o l - p y r i d i n e - o . r N HC1 (5:3:2 v/v). Paper electrophoresis was carried out with a horizontal type apparatus and paper chromatography by the ascending technique at room temperature. Reducing sugars were detected b y the alkaline silver nitrate spray 14, aminosugars b y the Elson-Morgan spray 15 or the ninhydrin spray and sialic acid b y the thiobarbituric acid spray le with a slight modification. Sulphate esters were detected b y dipping papers in an aqueous solution (o.5 % w/v) of trypaflavin and washing the papers with ethanol to remove excess trypaflavin. Spots of sulphate esters were stained in orange on yellow background. The detection of the spots is easier made under irradiation b y ultraviolet light: the spots appear dark on a fluorescent background. Authentic samples of N-acetyl- and N-glycolyl-neuraminic acids were gifts from Dr. T. YAMAKAWA. The other authentic sugars were commercial products. D E A E Sephadex and Sephadex were products of Pharmacia, Uppsala, Sweden. RESULTS
General properties of horatinsulphurie acid Horatinsulphuric acid is a general name given to the analogous heteropolysaccharide sulphates isolated from the liver of C. lampas. It is separated from neutral Biochim. Biophys. Acta, i o i (1965) 16-25
N E W P O L Y S A C C H A R I D E S U L P H A T E S FROM A G A S T R O P O D
21
polymers by electrophoresis on GEON (Fig. 4). The mobilities of the sulphate ester fraction in several buffer systems of different pH's were similar to those of nucleic acids and the sulphate ester cannot be separated from nucleic acid by this procedure. It may be noted that the preparation has two major sulphate ester fractions of different electrophoretic mobilities and sialic acid is associated with both fractions. Precipitation of the acidic polymers by trypaflavin as described under EXPERIMENTAL PROCEDURES was more suitable for the large scale preparation. Further fractionation of horatinsulphuric acid was made by column chromatography using DEAESephadex. Single DEAE-Sephadex chromatography resulted in the fractionation of the original mixture into groups of sulphated polysaccharides which compositions were closely related to each other. After the second chromatography, several sulphated polysaccharides which appeared homogeneous on paper electrophoresis were obtained. The mobilities on paper electrophoresis of the horatinsulphuric acid fractions were similar to those of chondroitinsulphuric acid at pH 4.0 and 6.0.
~ 0.30 < 0.20
0.1C x
0
10 Distance f r o m origin (cm)
20
Fig. 4. S e p a r a t i o n o f h o r a t i n s u l p h u r i c acid b y zone-electrophoresis on G E O N . - - - © - - -, su]phate ester; - - - O - - , sia]ic acid; - - / k - - , hexose b y a n t h r o n e ; - - O - - , a b s o r b a n c y a t 26o m/~.
Constituents of horatinsulphuric acid The results of analyses of horatinsulphuric acid fractions obtained from the first DEAE-Sephadex column are summarized in Table I. As can be seen from Table I, 32-62~/o of the total weight of the sulphate ester fractions are found to be made up of neutral sugars, hexosamines and sulphate. The high content of nitrogen indicates the presence of peptid'e as a component of horatinsulphuric acid fractions. The paper chromatographic analysis of the hydrolysate showed the presence of a few ninhydrinreactive materials other than amino sugars.
Analysis of the sugar component The compositions of the sugar portions of the fractions obtained from the second column of DEAE-Sephadex, Fractions 3' and 4' (see Fig. 2), were examined by Biochim. Biophys. Acta,
i o i (1965) 16--25
22
S. I N O U E
TABLE
i
ANALYTICAL GRAPHY
DATA
FOR
HORATINSULPHURIC
ACID
FRACTIONS
Eluant
Yield N (mg) (%)
SO~(%)
Hexose (%)
Hexosamine (%)
BY
COLUMN
CHROMATO-
Sialic acid
(%)
o.oi M TrisHC1 ( p H 8.0) 48
6.4
31
23
5-5
I-5
0. 3 0. 4 0. 5 0. 7 i.o
6. 3 6. 5 5-4 5.5 5.7
ii io 12 16 24
t5 16 i8 iS 20
0.i 6. 4 7.2 LI.8 I 1.8
3.2 3.8 2.8 -.~ 2.1
M M M M M
OBTAINED
ON DEAE-SEPHADEX
NaC1 NaC1 NaC1 NaC1 NaC1
12 20 20 17 14
paper chromatography. The samples were hydrolysed in sealed tubes with I N HC1 at ioo ° for 18 h. The hydrolysates were subjected to paper chromatography on W h a t m a n No. 3MM in Solvents I and II. Six spots of reducing sugars were observed by alkaline silver nitrate spray in hydrolysates of both Fractions 3' and 4'- Identical chromatographic patterns were obtained for the hydrolysates of Fractions 3' and 4'. Therefore, the component sugars of both fractions are identical. The Rote values and the assignment to authentic sugars are given in Table II. For quantitative analysis of each sugar, hydrolysates of Fractions 3' and 4' were loaded onto Whatman No. 3MM as bands and after development of the chromatogram in Solvent I, each band of sugar was located by the aid of a guide strip and cut; the sugar was eluted with water. Appropriate blanks for paper were prepared. The sugar was determined on the basis of its reducing power. The molar ratios of the sugars in Fractions 3' and 4' are listed in Table III. All the sugars which were separated in this way were identified with the corresponding authentic sugars listed in Table I I I by means of paper chromatography in Solvent I and II. Sugars 5 and 6 gave positive reactions with the Elson-Morgan and ninhydrin spray reagents. It m a y be noted that the molar ratio of the sugars in Fraction 3' and Fraction 4' are slightly different. The components of the sugar portion of the other fractions have not yet been analysed quantitatively, but qualitatively they are similar to those of Fractions 3' and 4'. TABLE PAPER
Sugar
II CHROMATOGRAPHIC
IDENTIFICATION
Ra~c in
Corresponding
. . . .
SI $2 $3 S4 S5 $6
OF SUGARS
s~gar
Solvent I
Solvent I I
1.22 i.o 9 I .oo 0.90 o.73 0.64
1.45 i. 18 t .oo o. 87 o..58 o..54
Fucose Mannose Glucose Galactose Glucosamine Galactosalnine
Biochim. Biophys. Acta, i o i (1965) 16 25
IN HORATINSULPHURIC
ACID
23
N E W P O L Y S A C C H A R I D E S U L P H A T E S FROM A G A S T R O P O D TABLE
III
COMPOSITION OF SUGAR
Molar ratio
F r a c t i o n 3' F r a c t i o n 4'
Fucose
Mannose
Glucose
Galactose
Glucosamine
Galactosamine
2 2
I i
i i
2 2
i 2
i 2
Evidence for the presence of sialic acid and its identification The mild acid hydrolysis of horatinsulphuric acid released a substance which, after periodate oxidation, reacts with the thiobarbituric acid reagent to give a chromophore exhibiting the light absorbancy spectrum identical with that observed for authentic sialic acids on similar treatment. The rate of release of the substance from Fractions 3' and 4' on heating in o.I N HC1 at 80 ° is demonstrated in Fig. 5. As can be seen from Fig. 5, lOO% release of sialic acid was attained when 20-25% of the total reducing power appeared. Thus, sialic acid m a y be considered to be located at the terminal position of the polysaccharide chain as is the case with other glycoproteins. The hydrolysate of Fraction 4' in o.I N HC1 at 80 ° for 60 min was subjected to paper chromatography in Solvents I I I and IV and the mobility of sialic acid in the hydrolysate was compared with that of authentic sialic acids. As shown in Table IV, the mobility of sialic acid from horatinsulphuric acid was identical with that of Nglycolylneuraminic acid rather than that of N-acetylneuraminic acid. TABLE
RF
IV
VALUES
OF SIALIC ACIDS
Solvent I I I Sialic a c i d f r o m h o r a t i n sulphuric acid o,33 N - G l y c o l y l n e u r a m i n i c a c i d 0.33 N-Acetylneuraminic acid o.41
Solvent I V
o. IO o.Io o,13
Molecular weight of horatinsulphuric acid Horatinsulphuric acid is not dialysable through a cellophane membrane. I t is eluted as a broad peak between the peaks of serum albumin and NaC1 when subjected to filtration through Sephadex G-25. The result indicates that the molecular weight of each molecule of horatinsulphuric acid is a few thousands. This value corresponds to the value (5ooo) calculated from the analytical data obtained from the most purified sample on assumption that each molecule has a sialic acid residue, possibly at the terminal position.
Infrared spectroscopy The infrared spectrum of Fraction 4' is shown in Fig. 6. The bands at 123o cm -1 and 850 cm -1 are attributable to vibrations of symmetric S = O and axial C-O-S, respectively. Biochim. Biophys. ,4cta, i o i (1965) 1 6 - 2 5
24
S. I N O U E Fr,3 I
100
~
~--~r-4,
5O Fr,-4 ~
Fo-3' 0
2'0
0
l
~ ._
40 GO Time (rain)
//
l
180
Fig. 5. Rates of release of sialic acid and reducing group by hydrolysis of horatinsulphuric acid fractions with o . i N HC1 a t 8o °. @, sialic acid; 0 , reducing power.
2.5 100
3
4
5
i
i
i
Wavelength (~J) 6 7 ~
6
9
f
I
I
10 11 12 I
I
I
80
6O
14 t
I
100 80
~
,
j
.
K
,
~
6o 40
40 k
20 0 4000
20 0 .
3. 0 0.0
.
2 0. 0 0
' ' 14 'o 0 18' O0 ' 1600 Wave number (crn -1)
.
. . 1200
.
1 0 0. 0
.
8. 0 0
Fig. 6. Infrared absorption spectrum of horatinsulphuric acid, Fraction 4'.
DISCUSSION
Horatinsulphuric acid may be classified as belonging to the group designated as mucoprotein by MEYER17 from its carbohydrate content. The acidity which can be ascribed to its high sulphate content is a characteristic feature of horatinsulphuric acid. Until recently, sulphated polysaccharides with only simple structures were known. These contain one kind of sugar or a repeating unit consisting of two kinds of sugars. Recently some workers have come to the recognition of the presence of sulphated glycoprotein 18,19. However, the sulphate contents of glycoprotein reported by these workers are not as high as that of horatinsulphuric acid. A careful examination proved that the compound is not present in tissues other than the liver in this gastropod. No trace was present in the digestive tract. This fact excludes the idea that the compound is derived from the other source taken as food by the gastropod. As uronic acid cannot be detected in the preparations of horatinsulphuric acid, the Biochim. Biophvs. Acta, i o i ('~965) 1 6 - 2 5
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
25
possibility that the preparations are contaminated by the sulphated polymer of the type of chondroitin sulphate or heparin was excluded. The heterogeneity of horatinsulphuric acid may be of interest from the view point of the mode of biosynthesis of heteropolysaccharide of this type. The heterogeneity seems to arise from the sulphate content, sugar composition and molecular weight. The significance of a sulphated compound of this type in C. lampas is not known. Polysaccharide sulphatase is active 2 in this gastropod and one can develop a theory that sulphate ester is playing important roles. In this connexion, LLOYD AND LLOYD2° also reported the presence of sulphated polysaccharide in the viscera of a marine mollusc which can be a substrate for the sulphatase preparation from the same source. Preliminary studies concerning the incorporation of 85S042- into horatinsulphuric acid, and hydrolysis of horatinsulphuric acid by the sulphatase or carbohydrase (personal communication from T. MURAMATSUof this laboratory) present in the liver of C. lampas indicated that the polysaccharide is metabolically stable as is the case for most polysaccharides. This work also gave evidence that sialic acid is distributed in a species of gastropod.
ACKNOWLEDGEMENTS
I am greatly indebted to Professor F. EGAMI for his guidance and interest throughout this work. Thanks are due to Dr. T. FURUHASHI for the collection of C. lam#as and Seikagaku Kogyo Co. for support. The work is supported in part by a grant from the Ministry of Education. REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 2o
T. SODA AND C. HATTORI, Bull. Chem. Soc. Japan, 8 (1933) 65. N. TAKAHASHI AND F. EGAMI, Biochem. J., 80 (1961) 384 . S. SUZUKI, N. TAKAHASHI AND F. EGAMI, Biochim. Biophys. Acta, 24 (1957) 444S. SUZUKI, N. TAKAI-IASHI AND V. EGAMI, J. Biochem. Tokyo, 46 (1959) I. T. SODA AND F. EGAMI, Bull. Chem. Soc. Japan, 13 (1938) 652. S. II~OUE AND F. EGAMI, J. Biochem. Tokyo, 54 (1963) 557. M. W . WHITEHOUSE AND H. BOSTR6M, Biochem. Pharmacol., 7 (1961) 135. A. G. LLOYD, Biochem. J., 72 (1959) 133. M. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956 ) 350. T. A. SCOTT, JR. AND E. H. MELVIN, Anal. Chem., 25 (1953) 1656. L. SVENNERHOLM, Acta Soc. Med. Upsalien., 61 (1956) 287. D. AMINOF1L Biochem. J., 81 (1961) 384. J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. W. E. TREVELYAN, D. P. PROCTER AND J. S. HARRISON, Nature, 166 (195 o) 444. S. M. PARTRIDGE, Biochem. J., 42 (1948) 238. L. WARREN, Nature, 186 (196o) 237. K. MEYER, in H . W . COLE, Some Conjugated Proteins, R u t g e r s U n i v e r s i t y Press, New B r u n s w i c k , 1953, p. 64. P. W. KENT AND J. C. MARSDEN, Biochem. J., 87 (1963) 38 p. L. ROBERT AND Z. DlSCHE, Biochem. Biophys. Res. Commun., io (1963) 209. P. F. LLOYD AND K. O. LLOYD, Nature, 199 (1963) 287.
Biochim. Biophys. Acta, i o i (1965) i 6 - 2 5
BIOCHIMICAET BIOPHYSICAACTA
BBA 85034 ISOLATION OF N E W P O L Y S A C C H A R I D E SULPHATES FROM C H A R O N I A LAMPAS
SADAKO INOUE* Department of Biophysics and Biochemistry, Faculty of Scie~zce, University of Tokyo, Tokyo (Japa;z) (Received August 1tth, i964)
SUMMARY A sulphated heteropolysaccharide named horatinsulphuric acid has been isolated from a sulphatase-rich gastropod, Charonia lampas. Horatinsulphuric acid, a mixture of analogous polymers, is obtained by the method of trypaflavin precipitation of sulphate esters. Fractionation made by column chromatography using DEAESephadex separates the mixture into several fractions in which the sulphate content varies from IO to 3o%. The composition of the sugar portion consists of fucose, mannose, glucose, galactose, glucosamine, galactosamine and sialic acid.
INTRODUCTION Charonia lampas, a marine gastropod**, has been noted for its active sulphatasesl,2. The function of the sulphatases in this gastropod has been solved only incompletely3, a. An idea that there must be natural substrates for the sulphatases led to the isolation of a glycan sulphate named charoninsulphuric acid from the mucous gland of this gastropod 5. Recently tissues of C. lampas were re-examined to find any other natural sulphate esters and it was found that the liver contains at least two families of polysaccharide sulphates. One, having the properties of a sulphated heteropolysaccharide, was named horatinsulphuric acid and the other sulphate ester was found to consist of a hitherto unknown sugar. A sulphate ester fraction distinguishable from charoninsulphuric acid was also separated from the mucous gland. This paper describes the isolation and properties of horatinsulphuric acid. A preliminary account of this paper has already appeared 6. EXPERIMENTAL PROCEDURE
The liver of C. lampas was frozen in dry ice-ether immediately after dissection and kept in a deep freezer until needed. - -;-Present address: l)epartment of Biochemistry, Tohoku University, School of Medicine, Sendai (Japan). .... B6shfi-hora" in Japanese. The same species is frequently referred to as Charonia sauliae. 13iochim. Biophys. Acta, ioi (1905) 16 25
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
17
Isolation procedure Step I: Extraction and precipitation by ethanol. The standard procedure was a s follows: The frozen liver (from 12 animals; 600 g) was homogenized with 1.2 1 of water in a Waring blendor. The homogenate was boiled for 5 min and centrifuged at 4000 rev./min for 15 min after cooling. The slimy residue was discarded. To the supernatant was added I volume of ethanol and the mixture was left to precipitate overnight in the cold. The precipitate was collected by centrifugation and dissolved in 15o ml of water. The insoluble material was removed by high-speed centrifugation (IO ooo rev./min for 30 min). The fraction containing horatinsulphuric acid was reprecipitated with 2 volumes of ethanol from the clear solution. The precipitate was washed and dried by the usual method. Yield: IO g. Step 2: Precipitation with trypaflavin. The ethanolic precipitate, IO g, was dissolved in ioo ml of water and 5 ml of a lO% aqueous solution of trypaflavin (acriflavin, 3,6-diamino-Io-ifiethylacridinium chloride) was added. The resulting mixture was left overnight in the cold. The precipitate was collected and washed with water. The washing was repeated (usually 3 times wit h 5o-ml portions of water) until the washing water became almost colourless. The trypaflavin precipitate was dissolved in 300 ml of 5 M NaC1. When 2 volumes of ethanol were added to the solution, horatinsulphuric acid was precipitated with a large amount of NaC1. The precipitate was collected on a sintered glass funnel and washed with ethanol to remove trypaflavin. The washed precipitate was dissolved in 600 ml of water and dialysed against running tap water. The dialysed solution was concentrated on an evaporator to 60 ml at a temperature below 4 °o . Four volumes of ethanol were added to the concentrated solution and the precipitate was collected, washed and dried by the usual method. Yield: 9o0 mg (crude sodium horatinsulphate). The main contaminant of the crude material was nucleic acid, for the latter also gave a precipitate with trypaflavin. The amount of nucleic acid in this preparation was about 30 % when calculated as RNA b y absorbancy at 260 m/,. Step 3: Column chromatography on DEAE-Sephadex. A column (I.O x 20 cm) was prepared with DEAE-Sephadex A-5o, coarse grade, and was washed thoroughly with o.oi M Tris-HC1 buffer (pH 8.0). The crude horatinsulphuric acid (292 rag), dissolved in 5 ml of the buffer was applied to the column and eluted with the buffer. 5-ml fractions were collected at a flow rate of I ml per min. Elutions wer~ successively performed with the buffer containing NaC1 of increasing concentrations. The eluate was analysed for sulphate ester, sialic acid, and ultraviolet-absorbing material b y the method described below. The elution diagram is shown in Fig. I. The fractions in every peak were pooled, concentrated to one-tenth volume b y a rotary evaporator, dialysed and lyophilised. The recovery of ester sulphate was 73 %. Step 4: Re-chromatography by gradient-elution technique. The fractions obtained b y column chromatography on DEAE-Sephadex were further purified b y a second chromatography on a column of DEAE-Sephadex, I.O x 15 cm. A concentration gradient of NaC1 was obtained b y introducing 500 ml of 0.5 M NaC1 into a mixing chamber containing 20o ml of o.oi M Tris-HC1 (pH 8.0). When the 0.5 M NaC1 was used up, 0.75 M NaC1 was added to the reservoir, and 4-ml fractions were collected. Typical elution diagrams obtained from the chromatography of 0.4 M and o.5 M NaC1 eluates from the first column are shown in Fig. 2a and Fig. 2b, respectively. Fractions Biochim. Biophys. Acta, i o i (1965) 16-25
i8
S. I N O U E Fr - I
0.15
Fr-3
Fr-6
0.10
.Q
< o.o~
o
r
50
0
t
Buffer
t
0.1MNaCI
t
0.2MNaCI
100
Fraction number
t
0.3MNaCI
t
OAMNaCI
f
150
0.SMNoCI
20q
t
0.7MNaCI
1MNaCI
Fig. I. F r a c t i o n a t i o n o f h o r a t i n s u l p h u r i c a c i d b y c o l u m n c h r o m a t o g r a p h y o n D E A E - S e p h a d e x . - - - © - - -, s u l p h a t e e s t e r ; D--~, s i a l i c a c i d ; - - © , a b s o r b a n c y a t 260 ml~.
obtained by chromatography on the second column were virtually free from ultraviolet-absorbing material and no trace of ribose was detected on paper chromatograms when the hydrolysates were examined.
Zone electrophoresis on GEON GEON (No. 237 S, Japan GEON Co.) was previously washed with dilute alkali, water and finally with the buffer used for the electrophoresis. The suspension of GEON in the buffer was poured into a plastic trough, 1.5 × 5.0 × 45 cm, placed horizontally. 0.05 M Tris-acetate (pH 6.0) was found to be most satisfactory for the procedure. 0.5 g of the 50% ethanolic precipitate of the liver extract was dissolved in 4.0 ml of the buffer, mixed with dry GEON powder and the resulting paste was poured in a gap of I cm width which was made across the GEON bed at a distance of IO cm from the end of the trough near the cathode. The electrophoresis was carried out for 7 h at 16-18 mA and i . i kV in the cold. After the electrophoresis, the bed of GEON was cut into sections of i cm each in width and each section was eluted with 20 ml of water. Portions of the eluate were examined for the presence of sulphate ester by trypaflavin, ultraviolet-absorbing material, sialic acid and hexose.
Semi-quantitative determination of sulphate ester Although there was a report 7 in which the complex formation between sulphate ester and acridine derivative was used in the estimation of sulphate ester, the method is not commonly used. In the present work, a simple method based on the same idea was effectively used in the detection and semi-quantitative determination of sulphate ester in the eluates from the columns or in the fractions separated by electrophoresis. The procedure is as follows: To 0.5 ml of the solution of the sample containing sulphate ester at a concentration below I/~mole, o. I ml of a 0.5 % aqueous solution of Biochim. Biophys. Acta,
i o i (1965) 1 6 - 2 5
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
°4° F
1o
o.3o~
~ ~t
/
',
Fr-3'
1/\)
o.2ol-
? ~ ~
I
"/ 0
19
20
40
A
,o
÷,~',o\
~
I/?:i... IE:-
60 Fraction number Fr-4'
0.30
F~ 1.0
[cl~ ~' 0.20
~ <
%~
I
,............................. 3.5
0.10
0
0
20
40
60 80 100 120 140 160 180 Fraction number Fig. 2. R e - c h r o m a t o g r a p h y of h o r a t i n s u l p h u r i c acid fractions : a, F r a c t i o n 3 of Fig. i ; b, F r a c t i o n 4 of Fig. i. - - - O - - - , s u l p h a t e ester; - - Q - - , sialic acid; - - ~ - - , hexose b y a n t h r o n e ; - - O - - , a b s o r b a n c y at 260 m/u; . . . . . . . concentration of NaC1.
trypaflavin was added. After 15 min, 2.5 ml of water were added and the turbidity which formed was measured at 660 m/~. As shown in Fig. 3, a linear~relationship between the turbidity and the concentration of sulphate ester was o~tained. The stability of the turbidity was influenced by the concentration of salt in the solution in which the sulphate ester was dissolved. Therefore, when the concentration of salt in the solution of the sample is high, the standard curve must be prepared in the presence of salt at a similar concentration. The measurement shown in Fig. 3 was made in the absence of salt. The method gave satisfactory reproducibility and the variation in the turbidity obtained in solutions of the same concentration was small from one experiment to the other.
Other analytical methods Sulphate was determined by the method of LLOYDs. Neutral sugars were determined by either the phenol-I-I2SO4 method 9 or the anthrone method 1°. A mixture of glucose, galactose and mannose (1:2:1) was used as a standard in the Biochim. Biophys. Acta, IOI (I965) 16-25
2o
s. 1NOUE
0.40
0.30
~0.20
0.10
0
0
I 0.5
mg
1.0
Fig. 3. Determination curve of horatinsulphuric acid by trypaflavin precipitation. The sulphate content of the horatinsulphuric acid preparation is 1.53/,moles SO~- per mg.
phenol-H2SO a reaction; Hexosamine was determined by a modified Elson-Morgan reaction u ; Sialic acid was determined by the thiobarbituric acid reaction according to AMINOFF'S procedurO2; Nitrogen content was estimated with the micro-Kjeldahl apparatus; Reducing power was determined by the method of PARK AND JOHZ~SON~S; Infrared spectra were measured in K B r discs with a Hitachi spectrophotometer.
Electrophoretic and chromatographic techniques Paper electrophoresis and paper chromatography were carried out on W h a t m a n No. 3MM or Toyo 5IA paper. The following buffer and solvent systems were used: Buffer I, o.o5 M Tris-acetate (pH 6.o); Buffer II, o.I M ammonium acetate-acetic acid (pH 4.o); Solvent I, ethylacetate-pyridine-water (2:1:2 v/v), upper phase; Solvent II, n-butanol-pyridine-water (6:4:3 v/v); Solvent I I I , n - b u t a n o l - n p r o p a n o l - o . i N HC1 ( i : 2 : i v / v ) ; Solvent IV, n - b u t a n o l - p y r i d i n e - o . r N HC1 (5:3:2 v/v). Paper electrophoresis was carried out with a horizontal type apparatus and paper chromatography by the ascending technique at room temperature. Reducing sugars were detected b y the alkaline silver nitrate spray 14, aminosugars b y the Elson-Morgan spray 15 or the ninhydrin spray and sialic acid b y the thiobarbituric acid spray le with a slight modification. Sulphate esters were detected b y dipping papers in an aqueous solution (o.5 % w/v) of trypaflavin and washing the papers with ethanol to remove excess trypaflavin. Spots of sulphate esters were stained in orange on yellow background. The detection of the spots is easier made under irradiation b y ultraviolet light: the spots appear dark on a fluorescent background. Authentic samples of N-acetyl- and N-glycolyl-neuraminic acids were gifts from Dr. T. YAMAKAWA. The other authentic sugars were commercial products. D E A E Sephadex and Sephadex were products of Pharmacia, Uppsala, Sweden. RESULTS
General properties of horatinsulphurie acid Horatinsulphuric acid is a general name given to the analogous heteropolysaccharide sulphates isolated from the liver of C. lampas. It is separated from neutral Biochim. Biophys. Acta, i o i (1965) 16-25
N E W P O L Y S A C C H A R I D E S U L P H A T E S FROM A G A S T R O P O D
21
polymers by electrophoresis on GEON (Fig. 4). The mobilities of the sulphate ester fraction in several buffer systems of different pH's were similar to those of nucleic acids and the sulphate ester cannot be separated from nucleic acid by this procedure. It may be noted that the preparation has two major sulphate ester fractions of different electrophoretic mobilities and sialic acid is associated with both fractions. Precipitation of the acidic polymers by trypaflavin as described under EXPERIMENTAL PROCEDURES was more suitable for the large scale preparation. Further fractionation of horatinsulphuric acid was made by column chromatography using DEAESephadex. Single DEAE-Sephadex chromatography resulted in the fractionation of the original mixture into groups of sulphated polysaccharides which compositions were closely related to each other. After the second chromatography, several sulphated polysaccharides which appeared homogeneous on paper electrophoresis were obtained. The mobilities on paper electrophoresis of the horatinsulphuric acid fractions were similar to those of chondroitinsulphuric acid at pH 4.0 and 6.0.
~ 0.30 < 0.20
0.1C x
0
10 Distance f r o m origin (cm)
20
Fig. 4. S e p a r a t i o n o f h o r a t i n s u l p h u r i c acid b y zone-electrophoresis on G E O N . - - - © - - -, su]phate ester; - - - O - - , sia]ic acid; - - / k - - , hexose b y a n t h r o n e ; - - O - - , a b s o r b a n c y a t 26o m/~.
Constituents of horatinsulphuric acid The results of analyses of horatinsulphuric acid fractions obtained from the first DEAE-Sephadex column are summarized in Table I. As can be seen from Table I, 32-62~/o of the total weight of the sulphate ester fractions are found to be made up of neutral sugars, hexosamines and sulphate. The high content of nitrogen indicates the presence of peptid'e as a component of horatinsulphuric acid fractions. The paper chromatographic analysis of the hydrolysate showed the presence of a few ninhydrinreactive materials other than amino sugars.
Analysis of the sugar component The compositions of the sugar portions of the fractions obtained from the second column of DEAE-Sephadex, Fractions 3' and 4' (see Fig. 2), were examined by Biochim. Biophys. Acta,
i o i (1965) 16--25
22
S. I N O U E
TABLE
i
ANALYTICAL GRAPHY
DATA
FOR
HORATINSULPHURIC
ACID
FRACTIONS
Eluant
Yield N (mg) (%)
SO~(%)
Hexose (%)
Hexosamine (%)
BY
COLUMN
CHROMATO-
Sialic acid
(%)
o.oi M TrisHC1 ( p H 8.0) 48
6.4
31
23
5-5
I-5
0. 3 0. 4 0. 5 0. 7 i.o
6. 3 6. 5 5-4 5.5 5.7
ii io 12 16 24
t5 16 i8 iS 20
0.i 6. 4 7.2 LI.8 I 1.8
3.2 3.8 2.8 -.~ 2.1
M M M M M
OBTAINED
ON DEAE-SEPHADEX
NaC1 NaC1 NaC1 NaC1 NaC1
12 20 20 17 14
paper chromatography. The samples were hydrolysed in sealed tubes with I N HC1 at ioo ° for 18 h. The hydrolysates were subjected to paper chromatography on W h a t m a n No. 3MM in Solvents I and II. Six spots of reducing sugars were observed by alkaline silver nitrate spray in hydrolysates of both Fractions 3' and 4'- Identical chromatographic patterns were obtained for the hydrolysates of Fractions 3' and 4'. Therefore, the component sugars of both fractions are identical. The Rote values and the assignment to authentic sugars are given in Table II. For quantitative analysis of each sugar, hydrolysates of Fractions 3' and 4' were loaded onto Whatman No. 3MM as bands and after development of the chromatogram in Solvent I, each band of sugar was located by the aid of a guide strip and cut; the sugar was eluted with water. Appropriate blanks for paper were prepared. The sugar was determined on the basis of its reducing power. The molar ratios of the sugars in Fractions 3' and 4' are listed in Table III. All the sugars which were separated in this way were identified with the corresponding authentic sugars listed in Table I I I by means of paper chromatography in Solvent I and II. Sugars 5 and 6 gave positive reactions with the Elson-Morgan and ninhydrin spray reagents. It m a y be noted that the molar ratio of the sugars in Fraction 3' and Fraction 4' are slightly different. The components of the sugar portion of the other fractions have not yet been analysed quantitatively, but qualitatively they are similar to those of Fractions 3' and 4'. TABLE PAPER
Sugar
II CHROMATOGRAPHIC
IDENTIFICATION
Ra~c in
Corresponding
. . . .
SI $2 $3 S4 S5 $6
OF SUGARS
s~gar
Solvent I
Solvent I I
1.22 i.o 9 I .oo 0.90 o.73 0.64
1.45 i. 18 t .oo o. 87 o..58 o..54
Fucose Mannose Glucose Galactose Glucosamine Galactosalnine
Biochim. Biophys. Acta, i o i (1965) 16 25
IN HORATINSULPHURIC
ACID
23
N E W P O L Y S A C C H A R I D E S U L P H A T E S FROM A G A S T R O P O D TABLE
III
COMPOSITION OF SUGAR
Molar ratio
F r a c t i o n 3' F r a c t i o n 4'
Fucose
Mannose
Glucose
Galactose
Glucosamine
Galactosamine
2 2
I i
i i
2 2
i 2
i 2
Evidence for the presence of sialic acid and its identification The mild acid hydrolysis of horatinsulphuric acid released a substance which, after periodate oxidation, reacts with the thiobarbituric acid reagent to give a chromophore exhibiting the light absorbancy spectrum identical with that observed for authentic sialic acids on similar treatment. The rate of release of the substance from Fractions 3' and 4' on heating in o.I N HC1 at 80 ° is demonstrated in Fig. 5. As can be seen from Fig. 5, lOO% release of sialic acid was attained when 20-25% of the total reducing power appeared. Thus, sialic acid m a y be considered to be located at the terminal position of the polysaccharide chain as is the case with other glycoproteins. The hydrolysate of Fraction 4' in o.I N HC1 at 80 ° for 60 min was subjected to paper chromatography in Solvents I I I and IV and the mobility of sialic acid in the hydrolysate was compared with that of authentic sialic acids. As shown in Table IV, the mobility of sialic acid from horatinsulphuric acid was identical with that of Nglycolylneuraminic acid rather than that of N-acetylneuraminic acid. TABLE
RF
IV
VALUES
OF SIALIC ACIDS
Solvent I I I Sialic a c i d f r o m h o r a t i n sulphuric acid o,33 N - G l y c o l y l n e u r a m i n i c a c i d 0.33 N-Acetylneuraminic acid o.41
Solvent I V
o. IO o.Io o,13
Molecular weight of horatinsulphuric acid Horatinsulphuric acid is not dialysable through a cellophane membrane. I t is eluted as a broad peak between the peaks of serum albumin and NaC1 when subjected to filtration through Sephadex G-25. The result indicates that the molecular weight of each molecule of horatinsulphuric acid is a few thousands. This value corresponds to the value (5ooo) calculated from the analytical data obtained from the most purified sample on assumption that each molecule has a sialic acid residue, possibly at the terminal position.
Infrared spectroscopy The infrared spectrum of Fraction 4' is shown in Fig. 6. The bands at 123o cm -1 and 850 cm -1 are attributable to vibrations of symmetric S = O and axial C-O-S, respectively. Biochim. Biophys. ,4cta, i o i (1965) 1 6 - 2 5
24
S. I N O U E Fr,3 I
100
~
~--~r-4,
5O Fr,-4 ~
Fo-3' 0
2'0
0
l
~ ._
40 GO Time (rain)
//
l
180
Fig. 5. Rates of release of sialic acid and reducing group by hydrolysis of horatinsulphuric acid fractions with o . i N HC1 a t 8o °. @, sialic acid; 0 , reducing power.
2.5 100
3
4
5
i
i
i
Wavelength (~J) 6 7 ~
6
9
f
I
I
10 11 12 I
I
I
80
6O
14 t
I
100 80
~
,
j
.
K
,
~
6o 40
40 k
20 0 4000
20 0 .
3. 0 0.0
.
2 0. 0 0
' ' 14 'o 0 18' O0 ' 1600 Wave number (crn -1)
.
. . 1200
.
1 0 0. 0
.
8. 0 0
Fig. 6. Infrared absorption spectrum of horatinsulphuric acid, Fraction 4'.
DISCUSSION
Horatinsulphuric acid may be classified as belonging to the group designated as mucoprotein by MEYER17 from its carbohydrate content. The acidity which can be ascribed to its high sulphate content is a characteristic feature of horatinsulphuric acid. Until recently, sulphated polysaccharides with only simple structures were known. These contain one kind of sugar or a repeating unit consisting of two kinds of sugars. Recently some workers have come to the recognition of the presence of sulphated glycoprotein 18,19. However, the sulphate contents of glycoprotein reported by these workers are not as high as that of horatinsulphuric acid. A careful examination proved that the compound is not present in tissues other than the liver in this gastropod. No trace was present in the digestive tract. This fact excludes the idea that the compound is derived from the other source taken as food by the gastropod. As uronic acid cannot be detected in the preparations of horatinsulphuric acid, the Biochim. Biophvs. Acta, i o i ('~965) 1 6 - 2 5
NEW POLYSACCHARIDE SULPHATES FROM A GASTROPOD
25
possibility that the preparations are contaminated by the sulphated polymer of the type of chondroitin sulphate or heparin was excluded. The heterogeneity of horatinsulphuric acid may be of interest from the view point of the mode of biosynthesis of heteropolysaccharide of this type. The heterogeneity seems to arise from the sulphate content, sugar composition and molecular weight. The significance of a sulphated compound of this type in C. lampas is not known. Polysaccharide sulphatase is active 2 in this gastropod and one can develop a theory that sulphate ester is playing important roles. In this connexion, LLOYD AND LLOYD2° also reported the presence of sulphated polysaccharide in the viscera of a marine mollusc which can be a substrate for the sulphatase preparation from the same source. Preliminary studies concerning the incorporation of 85S042- into horatinsulphuric acid, and hydrolysis of horatinsulphuric acid by the sulphatase or carbohydrase (personal communication from T. MURAMATSUof this laboratory) present in the liver of C. lampas indicated that the polysaccharide is metabolically stable as is the case for most polysaccharides. This work also gave evidence that sialic acid is distributed in a species of gastropod.
ACKNOWLEDGEMENTS
I am greatly indebted to Professor F. EGAMI for his guidance and interest throughout this work. Thanks are due to Dr. T. FURUHASHI for the collection of C. lam#as and Seikagaku Kogyo Co. for support. The work is supported in part by a grant from the Ministry of Education. REFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 2o
T. SODA AND C. HATTORI, Bull. Chem. Soc. Japan, 8 (1933) 65. N. TAKAHASHI AND F. EGAMI, Biochem. J., 80 (1961) 384 . S. SUZUKI, N. TAKAHASHI AND F. EGAMI, Biochim. Biophys. Acta, 24 (1957) 444S. SUZUKI, N. TAKAI-IASHI AND V. EGAMI, J. Biochem. Tokyo, 46 (1959) I. T. SODA AND F. EGAMI, Bull. Chem. Soc. Japan, 13 (1938) 652. S. II~OUE AND F. EGAMI, J. Biochem. Tokyo, 54 (1963) 557. M. W . WHITEHOUSE AND H. BOSTR6M, Biochem. Pharmacol., 7 (1961) 135. A. G. LLOYD, Biochem. J., 72 (1959) 133. M. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956 ) 350. T. A. SCOTT, JR. AND E. H. MELVIN, Anal. Chem., 25 (1953) 1656. L. SVENNERHOLM, Acta Soc. Med. Upsalien., 61 (1956) 287. D. AMINOF1L Biochem. J., 81 (1961) 384. J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. W. E. TREVELYAN, D. P. PROCTER AND J. S. HARRISON, Nature, 166 (195 o) 444. S. M. PARTRIDGE, Biochem. J., 42 (1948) 238. L. WARREN, Nature, 186 (196o) 237. K. MEYER, in H . W . COLE, Some Conjugated Proteins, R u t g e r s U n i v e r s i t y Press, New B r u n s w i c k , 1953, p. 64. P. W. KENT AND J. C. MARSDEN, Biochem. J., 87 (1963) 38 p. L. ROBERT AND Z. DlSCHE, Biochem. Biophys. Res. Commun., io (1963) 209. P. F. LLOYD AND K. O. LLOYD, Nature, 199 (1963) 287.
Biochim. Biophys. Acta, i o i (1965) i 6 - 2 5