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Further observations on the glycoproteins in human ovarian cyst fluids
3OO
BIOCHIMICAET BIOPHYSICAACTA
BBA 85065 F U R T H E R OBSERVATIONS ON T H E G L Y C O P R O T E I N S IN HUMAN OVARIAN CYST F L U I D S
J. R. DUNSTONE" AND Vq. T. J. MORGAN The Lister [nstit*tte of Preventive Medicine, London (Great Britain)
(Received August I6th, 1965)
SUMMARY An examination has been made of a number of ovarian cyst fluid glycoproteins that are sparingly soluble in water but are similar in general composition to the readily soluble blood-group specific glycoproteins obtained from the same source. Ultracentrifugal analysis of approx, o.i % w/v solutions of the sparingly soluble materials indicate that they have much larger sedimentation coefficients (s2o,w) and molecular weights than have the water-soluble glycoproteins. The sparingly soluble substances have more total amino acids and proportionally more aspartic and glutamic acids and cystine and less serine and threonine than have the soluble glycoproteins. The sparingly soluble substances mostly dissolve in buffered (pH 6.8-7.0, 0.05 M) solutions of thioglycolate, sulphite or cysteine, reagents that bring about the scission o f - S - S bonds. The behaviour and properties of the sparingly soluble glycoproteins suggest that they arise through the building up of relatively small glycoproteins to larger macromolecules through the formation o f - S - S - intermolecular associations. A number of ways whereby the formation of these sparingly soluble glycoproteins could occur are discussed.
INTRODUCTION Investigations on the chemical structure and immunological properties of human blood-group specific glycoproteins have mostly been made on the phenolinsoluble, water-soluble materials remaining after extraction of the freeze-dried contents of pseudomucinous ovarian cysts with cold 9o-95% w/w phenol 1-~. This method of preparation gives water-soluble group specific glycoproteins that have been well characterized. In most instances chemically similar glycoproteins that are sparingly soluble in water, and about which little is known, are also obtained. Digestion of the sparingly soluble material with pepsin at p H 2, or treatment b y ultrasonic irradiation, gives serologically active blood-group specific glycoproteins that are Present address: Department of Physical Biochemistry, The Australian National University, The John Curtin School of Medical Research, Canberra, A.C.T., Australia. Biochim. Biophys. Aela, ioi (1965) 3oo-314
OVARIANCYST GLYCOPROTEINS
301
readily soluble in water. However, with both methods of solubilization there is some uncertainty about the nature of the changes and extent of the degradation induced. It seems probable, however, that the glycoprotein that is sparingly soluble in water is a very large macromolecule that is easily changed by the cleavage of a few bonds into substances of smaller size which are readily soluble in water. The results of some preliminary attempts to obtain further information about the origin and nature of the sparingly soluble gel-like glycoproteins are given in the present paper. EXPERIMENTAL
Ovarian cyst glycoproteins. The glycoproteins are isolated from the freeze-dried contents of human pseudomucinous ovarian cyst adenoma by extraction at room temperature with 95 % w/w "liquid" phenol 4. The material remaining insoluble after thorough extraction with phenol is washed with a small amount of ethanol to remove excess phenol, suspended in water and dialyzed in Visking tubing. After dialysis is complete the viscous, gel-like, opalescent suspension is separated into water-insoluble and water-soluble components by centrifugation. The water-insoluble material is washed repeatedly with water and recovered by centrifugation at 30 ooo × g for 3 h. These glycoprotein preparations, which are mostly soluble to the extent of forming very dilute solutions (at most o. I °/o w/v) in water, are those used in the experiments described in this paper. The glycoprotein remaining in solution after dialysis of the phenol-insoluble preparation described above is the water-soluble specific blood-group substance. Sedimentation. Sedimentation-velocity experiments were performed in a Spinco Model E ultracentrifuge at 50 740 rev./min using the An-E rotor and sedimentation coefficients (expressed as Svedberg units: S) were corrected to water at 20 °. The runs were made at 25 ° and were controlled by the rotor temperature indication control unit. The partial specific volume of the substance was taken as 0.625, as reported by PUSZTAI AND ~V[ORGAN5. All measurements, except where stated otherwise, were made in 0.02 M KH2POa-K2HPO a buffer (pH 6.8) adjusted to 11 o.I with KC1. Chemical analyses. The preparations after drying from the frozen state were further dried over P205 for 30 min at 78° and 1-2 mm Hg pressure before analysis. The methods used for the determination of fucose 6 and for hexosamine 7 and reducing sugars s after acid hydrolysis were those used in earlier work. Nitrogen was determined by a micro-Kj eldahl procedure 9 followed by microdiffusion and titration in a standard Conway unit 1°. Sialic acid was determined by the method of WARREN11 after hydrolysis in o.I N H2SO4 for I h at 80 °. The carbohydrate content of solutions obtained directly from DEAE-cellulose columns was determined by the phenol-sulphuric method TM, while the amino acid content of these solutions was estimated by measuring the absorbance at 210 m/, (ref. 13). Serological activity. This was determined by the capacity of substances to inhibit in specific haemagglutination tests14,15. Glycoproteins possessing A, B, H and Le a activity le were investigated. Quanfitative amino acid analyses. These were carried out by the method of MOORE, SPACKMANAND STEIN17 as used by PUSZTAI AND •ORGAN is. In most experiments the glycoprotein was first oxidized with performic acid (16 h, --2 °) to convert cystine and/or cysteine to cysteic acid 19. Under these conditions certain amino acids Biochim. Biophys. Acta, IOI (1965) 3oo-314
302
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R. DUNSTONE, W. T. J. MORGAN
are extensively destroyed and quantitative amino acid analyses were therefore made on a few preparations before and after performic acid oxidation. The fully corrected 18 results for one preparation obtained from cyst fluid No. 485 are given in Table I. It is evident that decomposition of threonine, serine, leucine, phenylalanine and tyrosine occurred as a result of the performic acid oxidation. Corrections to compensate for the degradation of threonine and serine by performic acid have been applied to all analyses, but no corrections have been made for the losses of the other amino acids. Some typical analytical results are given in Table II. TABLE I EFFECT
OF PERFORMIC
ACID OXIDATION
ON T H E A M I N O A C I D C O M P O S I T I O N
OF BLOOD-GROUP
SPECIFIC
SUBSTANCES
Oxidized a n d n o n - o x i d i z e d s a m p l e s of m a t e r i a l s p a r i n g l y soluble in w a t e r from c y s t fluid No. 485 were h y d r o l y z e d w i t h 6 N HC1 a t 114 i i 7 ° for 24 h a n d a n a l y z e d q u a n t i t a t i v e l y for t h e i r cons t i t u e n t a m i n o acids. The r e s u l t s are e x p r e s s e d as t h e m e a n v a l u e s of t r i p l i c a t e d e t e r m i n a t i o n s a n d are recorded as g a m i n o a c i d / i o o g of s u b s t a n c e , a n d h a v e been c o r r e c t e d for d e g r a d a t i o n d u r i n g acid h y d r o l y s i s is.
A m i n o acid
Without oxidation 1.74 5-95 3.18 2.33 2.33 0.97 i .39 1.o6 1.51 o.8o
With oxidation
A s p a r t i c acid Threonine Serine G l u t a m i c acid Pr oline Glycin e Alanine C y s t i n e (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine
1.74 5.43 2.97 2.27 2.33 I .oi i .44 1.o 3 1.46 o.77
i ,2o
i .0 5
o. 73 0.78 --
o. i o o. I I
T o t a l a m i n o acids
23.97
21.88
o, 17
Complete amino analyses were not always carried out, as it appeared from earlier results 18 that sufficient data to allow the characterization of the amino acid moiety of blood-group specific glycoproteins could be obtained without the determination of the basic amino acids, lysine, histidine and arginine, which together make up less than IO% of the total amino acids. Column chromatography on DEA E-cellulose. Chromatography of dilute solutions of the glyeoproteins was carried out on ~5o cm × 2 cm columns of DEAE-cellulose, as already described ~°. The substance was eluted with NaC1 solution (continuous NaC1 gradient, usually o.oi M - I M), but in some experiments other eluants followed the NaC1 gradient in order to recover as much material as possible from the column. Unless otherwise stated 5o-ml fractions were collected at a rate of 50 ml/h and the elution patterns were determined by analyzing I-ml portions from each fraction. Solubilization experiments. The phosphate buffer (o.oi M KHo,PO a, o.oi M Biochim. t~iophys. Acla, i o i (1965) 3 o o - 3 i 4
303
O V A R I A N CYST G L Y C O P R O T E I N S TABLE
II
THE AMINO ACID COMPOSITION OF SOME SPARINGLY SOLUBLE AND THE CORRESPONDING WATER*SOLUBLE GLYCOPROTEINS FROM THE SAME OVARIAN CYST FLUID E x p e r i m e n t a l d e t a i l s a r e g i v e n in t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d a s (a) g o f a m i n o a c i d / i o o g o f s u b s t a n c e ; ( b ) / * m o l e s o f a m i n o a e i d / i o o / , m o l e s o f t o t a l a c i d s e s t i m a t e d . A n a l y t i c a l v a l u e s f o r m a t e r i a l s (i) s o l u b l e a n d (ii) s p a r i n g l y s o l u b l e i n w a t e r a r e g i v e n .
Amino acid Aspartic acid Threonine Serine Glutamicaeid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
A substance No. 485
B substance No. 413
H substance No. 476
Le a substance No. 35 °
(i)**
(i)*,'**
(i)*,*'"
(i)*'.***
(a) 0-75 5-19 3.03 1.16 1.74 0.6o 0.96 0.45 0.86 0.60 0.59 ---
15.93
(ii)
(ii)
(ii)
(ii)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
4 .0 31.2 20.6 5 .6 lO.8 5.7 7-7 2.7 5.2 3.3 3 .2 ----
7-3 21.6 15. 9 8.3 II.I 7.1 8.8 4.4 6. 3 3.3 4.5 0. 3 0. 5 0.6
0.79 3.80 1.98 0.73 2.00 0.58 1.39 o.ii 0.68 o.31 0.34 o.15 0.28 --
5 , i 1.82 27.4 3.97 16.1 2.44 4.3 2.11 14. 9 1.98 6.6 0.93 13. 4 1.14 0,8 0.96 5 .0 1.38 2.o o.91 2.2 1.35 0.8 - 1. 4 -o.31
8.2 2o.1 14.o 8.6 lO. 4 7.5 7.7 4.8 7 .1 4.2 6.2 --1.2
0.70 3.94 2.21 o.91 1.6o °.51 1.14 o.15 0.59 0.30 0.24 o.io 0.20 --
4.4 3.3 ° 31.6 4.65 19. 3 3.35 5.4 4 .26 12.1 2.58 5.9 1.23 i i . o 2.31 i . o 1.52 4-3 2.43 1.9 1.24 1.6 2.83 0. 5 0. 5 i . o 0.32 -0.55
9.5 15.o 12.2 ii.i 8.6 6.3 io.o 4.8 8.0 3.6 8. 3 0. 5 0. 7 1. 4
o.4o 3.66 1.69 0.59 1.66 0.55 1.o 5 o.ii 0.69 o.31 0.69 0.05 0.27 --
2.9 2.85 29.8 4.07 15.6 2.41 3.8 3.56 13.9 1.94 7.0 1.21 11. 3 2.00 0.9 1.14 5-7 1.88 2.2 0.80 5.1 2.20 0. 3 0.08 1. 5 - -o.15
13.14
19.3 °
12.59
3o.82
11.72
24.29
1.78 4.74 3.07 2-24 2.34 0.97 1-43 0.98 1.36 0.80 1.o9 0.09 o.15 o.17 21.11
* S o l u b l e in s a t u r a t e d ( N H a ) 2 S O a a t 6o °. ** I n s o l u b l e in s a t u r a t e d (iXTH4)2SO4 a t 6o °. "** C a l c u l a t e d f r o m d a t a o f PUSZTAI AND MORGAN 18.
K2HPO 4, o.o6 ~ KC1, p H 6.8) used in the solubilization experiments contained o.o5 M reducing agent, cysteine or sodium thioglycolate. The sulphite buffer was 0.048 M Na2HPO 4, 0.02 M KH2PO 4, 0.03 IM Na2SO 3, o.o13 M NaHSO z (pH 7.0). RESULTS
An examination of the solubility of the sparingly soluble glycoprotein preparations indicated that solutions of m a x i m u m concentration of about o.I °/o w/v could be prepared in most instances b y dispersing the geMike materials (about o.5% w/v) (a) in anhydrous formamide, followed by dialysis of the suspension against distilled water or buffer solutions at 2 ° and centrifuging at 30 ooo × g, I h or (b) in distilled water by stirring vigorously for up to 3 h and then removing undissolved substance by centrifuging (30 ooo × g, 3 h). The fact that small amounts of material continue to dissolve after repeated extraction of the centrifuge deposits with water indicates that the preparations have definite, but limited, solubility.
Ultracentrifugal examination Solutions were prepared, as described above, from sparingly soluble glycoprotein preparations obtained from cyst fluids No. 485 (group A) and No. 413 (group B). The ultracentrifugal patterns obtained with dilute solutions of both materials showed Biochim. Biophys. Acta,
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(b) lO.3 16.4 ii.o 11.6 8.1 7.7 lO.8 4-5 7.7 2.9 8.1 0.2 -0.5
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j. R. DUNSTONE, W. T. J. MORGAN
Fig. i. S e d i m e n t a t i o n d i a g r a m s of solutions p r e p a r e d from t h e s p a r i n g l y soluble glycoproteins in t h e presence, or absence, of thioglycolate. All p h o t o g r a p h s were t a k e n 24 m i n after r e a c h i n g a speed of 5 ° 74 ° r e v . / m i n ,The p h a s e plate angles were (a), (b) a n d (d), 45°; (e) a n d (f), 4o°; (c) 55°; (g) 6o °. D i a g r a m s (a), (b), (c) a n d (d) were o b t a i n e d w i t h m a t e r i a l s f r o m o v a r i a n cyst No. 485 . D i a g r a m s (e), (f) a n d (g) with m a t e r i a l s f r o m o v a r i a n c y s t No. 413 . T h e m e t h o d s of p r e p a r a t i o n of t h e v a r i o u s s a m p l e s are given in t h e text.
more than one sedimenting boundary (Fig. I a and e). The boundaries were not sharp, most probably owing to the low concentration and the relatively high degree of polydispersity. Account could not be taken of the Johnston-Ogston effect, nor of the effect of the concentration dependence of the sedimentation coefficients. The s%0,w values of the fastest sedimenting components were greater than 15 S and this considerably higher than those most frequently found (S°2o,w lO-12 S) for the upper molecular range of values determined for the soluble blood-group specific substances. This indicated that in these preparations were molecules with sizes much larger than those normally encountered in the water-soluble glycoproteins.
Chemical composition The qualitative and quantitative carbohydrate composition of the two waterinsoluble substances studied in the ultracentrifuge (Tables I I I and IV) indicated that in each instance the materials have similar compositions to those of the water-soluble glycoprotein obtained from the corresponding cyst fluid. Amino acid analyses revealed that each of a number of sparingly soluble glycoproteins had a greater amount of total amino acid than did the corresponding water-soluble glycoproteins and they contained a higher proportion of aspartic acid, glutamic acid and cystine and lower proportions of threonine, serine and proline (Table II). The presence of a greater proportion of cystine in the sparingly soluble preparations was confirmed after oxidizing each material with performic acid. A peak corresponding to that of cysteic acid replaced that of cystine and the amount of cysteic acid was found to be equivalent to that of the cystine previously found. The occurrence of relatively large amounts of cystine in the sparingly soluble materials suggested that their low solubility might be attributed to the formation Biochim. Biophys. Acla, IOX (1965) 3oo-314
O V A R I A N CYST G L Y C O P R O T E I N S TABLE
305
III
T H E COMPOSITION OF FRACTIONS O B T A I N E D FROM T H E S P A R I N G L Y SOLUBLE P R E P A R A T I O N FROM O V A R I A N C Y S T NO. 4 8 5 ( G R O U P A ) A F T E R R E D U C T I O N W I T H T H I O G L Y C O L A T E A N D C H R O M A T O G R A P H Y ON D E A E - c E L L U L O S E
E x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d a s (a) g o f a m i n o a c i d / i o o g o f s u b s t a n c e ; (b) p m o l e s o f a m i n o a c i d / i o o p m o l e s of the amino acids estimated. Other values a r e e x p r e s s e d a s g / i o o g o f s u b s t a n c e . F r a c t i o n s a s s h o w n in F i g . 2.
Original sz~bslance
W e i g h t (rag) Nitrogen Fucose Hexosamine Reducing sugar Sialic acid S e r o l o g i c a l a c t i v i t y (~,g)
272 6.3 15 25 45 3 .8 --
(a) Asparticacid Threonine Serine Glutamicacid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
Fraction numbers
1.78 4-74 3.07 2.24 2.34 0.97 1.43 o.98 1.36 0.80 1.°9 0.09 o.15 o.17 2 i. i i
I
2
3
4
5
43 5.5 16 27 58 3.4 0.2
85 5.6 15 25 51 4 0.25
19 5.7 iI 25 46 5.1 0.6
iI 4.2 7 16 48 2.7 0. 7
31 5.6 15 23 5i 3.6 0.2
(~) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
7.3 21.6 15.9 8. 3 ii.i 7.1 8.8 4.4 6.3 3.3 4-5 0. 3 0. 5 o.6
3-9 33 .0 21.8 5.1 8.9 6.7 8.9 2.0 4.7 2.5 1-9
1.17 4-4 ° 2.98 1.53 1.5o 0.85 1.12 o.71 1.o4 0.57 0.67
o.17
7.5 16.8 14.8 lO. 5 7.8 9.9 8.4 5-4 7.4 2.9 4.3 1.8 0.9 1.6
l°.3 15. 5 13.9 9.9 6. 5 13.2 9.7 5-9 6.2 3.8 3-9 --
o.6
5.9 24. 9 18.8 7.0 8.8 9.1 8.5 3.9 6.o 2.9 3.4 --o.8
o-75 5.74 3.35 1.io 1.49 0.74 1.15 o.36 o.80 0-47 o.37 --o.13 16-44
16.66
1.86 3.72 2.89 2.88 1.66 1.38 1.39 1.32 1.62 o.71 1.o5 0.60 0.28 o.43 21.69
2.o7 2.78 2.20 2.18 1.13 1.49 1.3o 1.o7 I.iO 0.75 0.78 ---o.27 17-12
1.2
1.84 4.24 2.5 ° 1.94 1.45 i.oi 1.4 ° 0.62 1.11 0.57 0.60 o.16 ---
(b) 9.1 23.3 15.6 8.7 8.2 8.8 lO.3 3.4 6.2 2.8 3.0 o.6 ---
17,44
of intermolecular disulphide bonds and through them the formation of macromolecular complexes of much larger size. To test this suggestion the solubility behaviour of some sparingly soluble glycoproteins was examined in dilute, neutral solutions of reducing agents, such as thioglycolate, cysteine and sulphite, that are known to bring about the scission of disulphide linkages. Preliminary experiments indicated that the glycoproteins sparingly soluble in water obtained from cyst fluids No. 485 (A) and 413 (B) dissolved readily in a phosphate-thioglycolate or phosphate-cysteine buffer at pH 6.8. The glycoprotein preparations obtained from cyst fluids No. 476 (H) and 35o (Lea) however were less readily soluble under similar conditions. Substance No. 476 dissolved to the extent of about 5o% in 2o h and dissolved almost completely after a longer time. After several weeks the reducing agents dissolved not more than about 2o% of substance No. 35o and thereafter the solubility remained unchanged. Solution of the substances in phosphate-sulphite buffer (pH 7.o) was more rapid, the glycoprotein materials No. 485 and 413 dissolving immediately and No. 476 in about Biochim. Biophys. Acla, i o i (1965) 3 o o - 3 1 4
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j.R.
D U N S T O N E , W. T. J. MORGAN
TABLE IV THE COMPOSITION OF FRACTIONS OBTAINED FROM THE SPARINGLY SOLUBLE PREPARATION FROM OVARIAN CYST NO. 413 (GROUP B) AFTER REDUCTION WITH THIOGLYCOLATE AND CHROMATOGRAPHY O N D E A E - C E L L U L O S E E x p e r i m e n t a l d e t a i l s a r e g i v e n in t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d as (a) g o f a m i n o a c i d / I o o g of s u b s t a n c e ; (b)/~moles of a m i n o a c i d / i o o / , m o l e s of the a m i n o acids e s t i m a t e d . O t h e r values are e x p r e s s e d as g / i o o g o f s u b s t a n c e . F r a c t i o n s as s h o w n in Fig. 3.
Original substance
Fraction numbers I
W e i g h t (rag) N Fucose Hexosanline Reducing sugar Sialic a c i d S e r o l o g i c a l a c t i v i t y (pg)
Aspartic acid Threonine Serine Glutamieacid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
300 5,9 13 25 49 6. i --
6 3.2 16 21 42 I. i 0.07
(a)
(b)
(a)
1.82 3.97 2.44 2.11 1.98 0.93 1.14 0.96 1.38 o.91 1.35 --
8.2 2o.1 14-o 8.6 lO. 4 7.5 7.7 4.8 7 -1 4.2 6.2
0.90 2.70 1.28 0.60 1.o 7 0.64 0.73 0.07 0.30 0.27 0.20
o.31
1.2
19.3 °
2
-8.76
3
20 4-3 17 21 45 1.7 0.08
(b) (a)
80 4.7 12 22 41 5-7 0.09
5
9 5 -1 ii N.D.* N.D. 5 .8 0. 4
(a)
(b)
1.49 3.56 2.52 1.72 1.37 0.87 1.24 0.63 0.88 0.53 0.78 0.25
8.1 1.53 21.6 3.32 17.3 2.57 8.5 1.77 8.6 1.6o 8.4 1.o4 i o . i 1.26 3.8 0.70 5.4 1 . ° 9 2.9 0.43 4.3 0.76 i . o o.17 -0.09 0.22
8.1 1.81 19.2 4.33 8.3 2.74 8. 3 2.38 9.6 1.66 9.5 1.26 9.8 1.44 4.0 o,66 6. 4 1,6o 2.3 o,78 4 .0 1,3o 0.6 - 0. 4 - i . o 1.3o
16.55
21.26
1,54 3,58 2,42 1,27 i,ii o.71 1-o9 0.26 0.69 0.35 0.37
9.6 25. 7 I9.1 7.2 8.0 7 .8 lO.2 1.8 4.9 2.2 2.3
--
0.22
1.2
-15.84
(a)
(b)
57 5 .8 9 21 37 5.5 0.2
(b)
8.7 28. 7 15.6 5.2 11. 9 lO.9 IO.5 o. 7 3-3 2.6 1.9
13.61
4
(a)
(b) 7.4 19-9 14.2 8.8 7.9 9.2 8.8 3.0 7.5 3.2 5.4 --4.7
" N.D. = Not determined.
36 h. Glycoprotein preparation No. 35o dissolved completely only after repeated treatment with freshly prepared sulphite. The ease with which preparations No. 485 and 413 dissolved in the reducing agents suggested that their behaviour in buffered o.o5 M thioglycolate (pH 6.8) should be examined more closely. Portions of the substances were therefore dispersed (about o.5% w/v) in anhydrous formamide as described earlier and solutions of the soluble fractions in water recovered. Portions of these solutions (approx. o.1% w/v) from each of the glycoproteins were dialysed against (a) phosphate buffer and (b) phosphate -thioglycolate buffer. The dialysed solutions were then examined in the ultracentrifuge. The fastest sedimenting boundary in the non-reduced substance No. 485 ( F i g . I a ) disappeared after reduction (Fig. Ib). Longer centrifuging caused the latter peak to separate into two components with S%o,w values of about IO S and 8.5 S. Similar changes were produced under the same conditions by the reduction of a dilute Biochim. Biophys. Acta,
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OVARIAN CYST GLYCOPROTEINS
solution of glycoprotein No. 413, but they were less marked. Nevertheless differences in the relative proportions of the components after reduction were observed: Fig. ie, f. Attempts to reverse the dissociation were made by removing the reducing agent from preparation No. 485 by dialysis against oxygenated water at 2 ° for several days. No changes in the ultracentrifuge pattern were produced by this treatment (Fig. Id), suggesting that none of the original and heavier material was reformed. Solutions prepared by dissolving the sparingly soluble glycoproteins in thioglycolate buffer without previous dispersion in formamide, were similarly investigated. More of the glycoproteins (No. 485 and 413) dissolved than after treatment with formamide and the ultracentrifugal patterns obtained indicated that the fastest moving components (Fig. Ia and e) before reduction were not detected in the reduced preparations (Fig. ic and g). Longer centrifuging of preparation No. 485 (Fig. IC) showed it to be composed of two major components (s%0,w values about IO S and 8 S), but the material from preparation No. 413 sedimented as a single peak with the characteristics of a highly polydisperse material. The peak areas, when corrected for sectorial dilution, decreased in value as the run progresssed, indicating the probable continuous sedimentation of very large macromolecules. B e h a v i o u r on D E A E - c e l l u l o s e columns
The behaviour of the solubilized glycoproteins on columns of DEAE-cellulose was examined. The sparingly soluble glycoproteins, about 300 mg of each, were dis1.2F 1 4 - - 1 - - 4 ~ r - - ~ . - 2 ~ 3
~:~
4
/
!
1.0-
0.8
0.6 u £1
1.0
m
< 0.4
0.8
0.2
3.4~
Z
0.2 ~. 0
20
40 Tube number
60
80
100
Fig. 2. C h r o m a t o g r a p h y on D E A E - c e l l u l o s e of s o l u t i o n s of s p a r i n g l y soluble g l y c o p r o t e i n o b t a i n e d f r o m o v a r i a n c y s t fluid No. 485 (group A). D e t a i l s of t h e p r e p a r a t i o n are g i v e n in t h e t e x t . Cont i n u o u s g r a d i e n t e l u t i o n w i t h NaC1 is s h o w n b y t h e line . . . . . . C o n t e n t s of tubes, each c o n t a i n i n g 50 m l were pooled as s h o w n b e t w e e n a r r o w s to give t h e v a r i o u s fractions. 0 - - - 0 , a b s o r b a n c e a t 21o m ~ ; Q)--C), a b s o r b a n c e a t 485 m/z ( p h e n o i - s u l p h u r i c a c i d method13).
Biochim. Biophys. Acta, i o i (1965) 3oo-314
308
j. R. DUNSTONE, W. T. J. MORGAN 14-1 ~
2
~4~
3
~
4
1.2
1.Q
0.8
0.6
1.0 ou} ~" 0.4
/
-0.8
/
// /
0.2
J
-o.6~
/ -0,4~
0.2 O
0
2o
40
6o
U
80
Tube number"
Fig. 3. C h r o m a t o g r a p h y on DEAE-cellulose of solutions prepared from the sparingly soluble p r e p a r a t i o n from ovarian cyst fluid No. 413 (group B). Solutions, obtained by treating 3oo m g portions of material with (a) thioglycolate and with (b) water, as described in the text, were applied to the column. Continuous gradient elution with NaC1 is s h o w n by the line . . . . . . Contents of tubes, each containing 5o ml, were pooled as s h o w n between the arrows to give the various fractions. Absorbance at 485 mff (phenol-sulphuric acid m e t h o d TM) for (a) O - - Q and (b) O - - O .
solved in thioglycolate-containing buffer (5o ml, p H 6.8), dialysed against distilled water to give about o.5% w/v solutions which were then made o.oi N with respect to NaC1 and applied to a DEAE-cellulose column (15o cm × 2 cm). The elution patterns obtained for preparations No. 485 and 413 are given in Figs. 2 and 3, respectively. The values plotted in Fig. 2 are based on carbohydrate determination by the phenol-sulphuric acid method 12 and absorbance at 21o m# (ref. 13). The curves obtained for substance No. 485 (Fig. 2) indicated that there were no appreciable amounts of a substance composed only of amino acids. A similar result was obtained for substance No. 413 . Preparation No. 485 was resolved into several fractions. The first four were obtained by gradient elution with NaC1, the last (Fraction 5) b y elution with o.2 M NaOH; this fraction emerged with the NaOH front. Analytical values obtained for these fractions are given in Table I I I . A small amount of material was eluted from the column when phosphate buffer (0.2 3/[ NaH~PO4-Na~HPO4, pH 6.8) was used as eluant immediately after the NaC1 gradient was complete and before elution with NaOH started, but because the fraction was not free from buffer ions, the analytical figures obtained are not included in Table I n . However, the following values obtained for the material eluted with phosphate buffer: aspartic acid, 12.o; threonine, 12.o; serine, 12.2; glutamic acid, 11.7; proline, 5.7; glycine, 15.4; alanine, 8.5; cystine Biochim. Biophys. Acta, IOI (1965) 3oo-314
OVARIAN CYST GLYCOPROTEINS
309
(half), 6. 7 (expressed as/,moles of amino acid/ioo/,moles total amino acids estimated), are of considerable interest when compared with the amino acid distribution normally obtained (Tables II, III) and especially when compared with that for the glycoprotein recovered in the last fraction to be eluted with NaC1 (Fraction 4). Fraction I (Fig. 2, Table III) closely resembled in chemical composition the water-soluble material obtained from the same cyst fluid (Table II). Fractions 2, 3 and 4 (Fig. 2, Table III) and the material eluted with phosphate buffer contained progressively larger proportions of aspartic acid, glutamic acid, cystine (half) and glycine and smaller proportions of threonine, serine and proline. Fraction 5 (Table III) resembled more closely in composition the original material. Distinct variations in the small amounts of sialic acid in the various fractions were also observed, and most probably influenced to some extent the elution behaviour of the fractions collected. The total amount of amino acid in each fraction was, with the exception of Fraction 3, Table III, smaller than the amino acid content of the original preparation. Fractions I and 2 (Table III) had similar ultracentrifugal patterns characterized by single "hypersharp" peaks with only a very small amount of faster sedimenting material and compared closely with those obtained for the water-soluble glycoprotein isolated from the same cyst fluid. The other fractions were considerably more heterogeneous and Fraction 5 contained two major components. All fractions were active in haemagglutination inhibition tests; Fractions I and 2 (Table III) were as active as the highly-purified, water-soluble preparation (Table II) from the same cyst fluid. The other fractions were less active. The glycoprotein No. 413 was similarly separated into four fractions by gradient NaC1 elution from a DEAE-cellulose column (Fig. 3). Again a last and fifth fraction was recovered by elution with 0.2 M NaOH. The analytical data for these fractions are given in Table IV. Each fraction contained a greater proportion of aspartic acid and glutamic acid (Table IV) than did the corresponding water-soluble material (Table II). The proportions of threonine and serine in Fractions I and 2 were similar to those of the water-soluble material; the other fractions contain somewhat less threonine. The proportion of cystine (half) was increased in the later fractions (Table IV, Fractions 3, 4 and 5). The first fraction, which contained the smallest amount of total amino acids, resembled more closely in Fattern the water-soluble preparation than did any of the other fractions. Fractions 3 and 5 were examined in the ultracentrifuge. The sedimentation diagrams indicated in each instance one maj or component with smaller but nevertheless significant amount of slower sedimenting material. All fractions were serologically active ; Fractions, I, 2 and 3 were as active, within the limits of the tests, as was the purified water-soluble blood-group substance obtained from the same cyst; Fractions 4 and 5 were less active. In order to make a valid interpretation of these results it was necessary to establish that the fractions obtained from the DEAE-cellulose columns were the materials solubilized by reduction of the sparingly soluble glycoprotein with thioglycolate and not soluble components already present in the interstices of the gel-like preparations. Portions (300 mg) of sparingly soluble substance No. 413 were suspended in distilled water (5o ml) and stirred vigorously for several days. The suspensions were then centrifuged (30 ooo × g, 3 h) and the supernatant solutions, suitably concentrated, were chromatographed on DEAE-cellulose columns. The total amount of soluble material was small compared with that obtained from the same glycoBiochim. Biophys. dcta, i o i (1965) 3oo-314
31o
j.R.
DUNSTONE, W. T. J. MORGAN
protein preparation after treatment with thioglycolate buffer. The elution patterns obtained for the material soluble in water and in thioglycolate buffer are given in Fig. 3 and indicate that the soluble substances recovered after the thioglycolate treatment could not be obtained by simple extraction with water and thus, were not soluble glycoproteins trapped in the interstices of the gel structure. The ready solubility of the sparingly soluble glycoprotein in reagents which specifically reduce disulphide bonds prompted an attempt to determine the amount of free sulphydryl groups in one preparation, after reduction with sulphite using p-chloromercuribenzoate. The sparingly soluble glycoprotein (IOO rag) obtained from cyst fluid No. 485 was suspended in 5.0 ml ofo.2 M acetate buffer (pH 5.3) containing 0.05 M Na2SO 3. After the material had dissolved 3.0 ml of 5 mM sodium p-chloromercuribenzoate was added and 16 h later a portion of the solution was titrated with 5 mM cysteine, using sodium nitroprusside as external indicator to determine the amount of p-chloromercuribenzoate unreacted with the free sulphydryl groups of the reduced glycoprotein. The glycoprotein contained 8.1/,moles of sulphur, calculated from the amount of cystine present (Table II). The results of the experiment showed that 3.6/*moles of sulphydryl sulphur were present after reduction. The scission of a disulphide bond with sulphite proceeds according to the equation: R-S-S-R
+ SOa '~- = R - S - + R S - S - O 3-
if all the sulphur in the original glycoprotein were present as disulphide sulphur, 4.05/,moles of sulphydryl sulphur should have been obtained; if incomplete reduction had occurred the value would be lower. On the other hand, if some of the sulphur of the original glycoprotein were present as sulphydryl sulphur the yield should be greater than 4.05/*moles. Therefore, it would appear that most of the sulphur in the glycoprotein is present as disulphide sulphur. An attempt to determine the free sulphydryl groups in the sparingly soluble preparation without previous reduction was unsuccessful because of the presence of the thick opalescent gel during this determination. Thus,
Digestion with pepsin The composition of the water-soluble glycoproteins obtained from the sparingly soluble gel-like preparation after digestion with pepsin was determined. Glycoproteins (300 nag) No. 485 and 413 were completely solubilized as described in Table V. The analytical figures for the substances before treatment with pepsin are given in Table I I. The first and largest fraction from each solubilized glycoprotein, at a concentration of about I °/o (w/v) in water, precipitated within the limits of 48 and 52% (v/v) ethanol, and in this property resembled closely the water-soluble blood-group substances. The analytical data for these fractions, obtained from glycoproteins No. 485 and No. 413 were N, 5.8 and 5.1; fucose, 17 and 14; total amino sugar, 27 and 25; total reducing sugars, 55 and 54, and sialic acid, 3.1 and 4.6, respectively. The amino acid composition in each instance (Table V) was closely similar to that of the corresponding water-soluble blood-group substance given in Table II except for a somewhat greater proportion of cystine (half). On examination in the ultracentrifuge both substances sedimented as single peaks, but were somewhat more polydisperse than the corresponding water-soluble glycoproteins. The smaller fractions precipitating at higher ethanol concentrations (Table V) Biochim. Biophys. Acta, i o i (1965) 3oo-314
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TABLE V CHEMICAL PROTEINS
COMPOSITION SPARINGLY
OF
THE
SOLUBLE
IN
WATER
SOLUBLE
WATER
AFTER
GLYCOPROTEINS TREATMENT
WITH
ISOLATED
FROM
THE
GLYCO-
PEPSIN
Portions of the sparingly soluble preparations (300 mg) were suspended in o.i M citric acidNaoHPO 4 buffer (pH 2.2) and incubated with crystalline ( x 2) pepsin (2 mg) at 37 ° for 16 h. The resulting solutions were dialysed and the glycoproteins isolated by repeated fractionation with ethanol. The materials were then dissolved in water, centrifuged at 3° ooo > g for I h and the s u p e r n a t a n t solutions dried from the frozen state. Amino acids are expressed as (a) g of amino acid/ioo g of substance and (b) /~moles of amino acid/ioo #moles of total amino acids estimated. A substance No. 485 Weight (mg) Ethanol fraction (v/v%)
z3o 48-52 (a)
Asparticacid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Methionine Total amino acids
23 52-59 (b)
(a)
39 59-75 (b)
0.86 4-5 1.38 5-7 5.8o 28.6 5.15 24.0 3.25 21.7 3.48 16.2 1.23 5.8 1.84 6.9 1.82 i i . i 2.48 11. 9 0.75 6.9 1.o7 8.0 1.o 3 8.0 1.53 9.5 0.64 3-7 1-o3 4.8 0.88 5.2 1.3o 6.1 o.41 2.2 o.61 2.6 0.49 2.3 0.78 3.3 -- - 0.27 I.O 17.16
2o.92
B substance No. 4z3
(a)
14o 48-52 (b)
(a)
32 52-59 (b)
(a)
2.09 8.2 1.o 3 5-9 2.o6 3.91 17.1 4.42 28.4 4-3 ° 2.84 14.1 2.29 16. 7 3.32 2.35 8.3 1.22 6. 4 2.81 2.53 11. 5 1.63 lO.8 2.80 1.26 8.8 0.74 7-5 1.63 1.37 8.0 1.17 io.o 1.59 1.82 7-9 0-47 3 .0 1.36 1.71 7 .6 0.87 5.7 1.41 0.79 3.1 0.48 2.8 0.64 1.36 5.4 0.48 2.8 0.97 0.64 22.o3
14.8o
23.53
3° 59-75 (b)
(a)
(b)
7.6 17.6 15. 5 9.3 11.9 lO-7 8.8 5.5 5.9 2. 4 3.6 1.2
2.88 5.59 2.64 4 .06 2.54 1-46 1.63 1.73 1.78 0.95 1.53 --
9.4 20-4 ii.o 12.o 9.6 8.5 8.0 6.2 6-7 3.1 5.1 --
26.79
s h o w e d g r e a t e r p r o p o r t i o n s o f c y s t i n e (half), a s p a r t i c a c i d a n d g l u t a m i c a c i d a n d l o w e r proportions of threonine and serine than did the first fractions and the corresponding w a t e r - s o l u b l e specific g l y c o p r o t e i n s ( T a b l e I I ) . T h e m a t e r i a l r e c o v e r e d f r o m t h e p e p s i n digests accounted for about two-thirds of the sparingly soluble material digested. DISCUSSION T h e g l y c o p r o t e i n s o c c u r r i n g i n o v a r i a n c y s t fluids t h a t h a v e b e e n m o s t t h o roughly examined have serological properties associated with blood-group specificity, are readily soluble in water and have molecular weights between 2-105 and 2.106 . I t is f r e q u e n t l y f o u n d h o w e v e r t h a t p a r t o f t h e v i s c o u s o p a l e s c e n t g l y c o p r o t e i n p r e p a rations obtained are sparingly soluble in water. These sparingly-soluble, gel-like materials resemble, in general chemical pattern and in certain physical properties, the soluble glycoproteins and can be readily solubilized with little change in composition by enzymic digestion or ultrasonic irradiation. The results now reported illustrate certain additional features that distinguish more clearly the sparingly soluble glycoproteins from the corresponding water-soluble p r e p a r a t i o n s o b t a i n e d f r o m t h e s a m e c y s t fluid. C h e m i c a l a n a l y s i s c o n f i r m s t h a t t h e s e substances are glycoproteins and their ultracentrifugal examination in dilute solution Biochim. Biophys. Acta, IOi (1965) 3oo-314
312
J.R. D U N S T O N E , W . T. j. M O R G A N
indicates that they have very much larger sedimentation coefficients than do the corresponding water-soluble substances. The results of more exact amino acid analyses also show that for the four sparingly soluble preparations examined, each has an amino acid pattern that is different in certain important respects from that of the corresponding water-soluble glycoprotein. The sparingly soluble preparations have an appreciably greater amount of total amino acid, a smaller proportion of threonine, serine and proline and a larger proportion of aspartic acid, glutamie acid and cystine than do the corresponding water-soluble glycoproteins. The properties of low solubility in water or buffers, of large molecular size, and of having enhanced proportions of cystine, suggest that the sparingly soluble glycoproteins are very large macromolecular species built up through the formation of a number of intermolecular disulphide (-S-S-) bonds. The demonstration that these sparingly soluble substances dissolve at neutral pH in simple reducing agents, such as thioglycolate and cysteine that are known to bring about cleavage of disulphide bonds, and that there is a simple relationship between the number of free - S H groups after sulphite reduction, and the total sulphur content, supports this suggestion. Some further evidence for this idea was obtained from ultracentrifugal analysis. The fastest sedimenting component in the original sparingly soluble preparations was absent, or considerably reduced in amount, when dilute solutions of these materials were treated with thioglycolate. The first and readily soluble fraction recovered from the sparingly soluble glycoprotein obtained from cyst No. 485, after solubilization in thioglycolate and separation on a DEAE-cellulose column, had an overall composition not unlike that of the water-soluble glycoprotein obtained from the same cyst fluid. Later fractions eluted with increasing salt concentration, however, had considerably more aspartic acid, glutamic acid and cystine than did the first fractions. The sparingly soluble glycoprotein obtained from cyst No. 413 after solubilization with thioglycolate and separation on a DEAE-cellulose column, also gave soluble components in which the proportions of threonine, serine and cystine were not dissimilar to those obtained for the water-soluble glycoprotein recovered from the same cyst fluid, but the early fractions contained a significantly higher proportion of aspartic and glutamic acids than did the glycoprotein originally soluble in water and obtained from the same cyst fluid. It is known however that homogeneous glycoprotein preparations are not necessarily obtained as a result of a single chromatography on DEAE-cellulose, as used in these experiments, and it is possible that the early fractions from the DEAE-cellulose column, while containing a relatively high proportion of glycoprotein similar in chemical composition to the water-soluble blood-group substances, remain contaminated with material that occurs more plentifully in the later fractions that are rich in acidic amino acids. The possibility of bringing about a more complete resolution by rechromatography was not investigated. The major water-soluble glycoprotein fractions obtained after digesting the sparingly soluble preparations with pepsin resembled the water-soluble blood-group substances in general chemical composition, but here again there was a significantly greater proportion of acidic amino acids and cystine in the solubilized product than in the corresponding soluble glycoprotein from the same cyst fluid. The fractions obtained by precipitating the digest with higher concentrations of ethanol however contained proportionally more acidic amino acids and cystine. Biochim. l~iophys. Acta, i o i ( i 9 6 5 ) 3 o o - 3 1 4
OVARIAN CYST GLYCOPROTEINS
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The fact that a considerable proportion of the sparingly soluble glycoprotein after solubilization with thioglycolate or sulphite can be recovered as a readily soluble glycoprotein that is relatively poor in aspartic and glutamic acids and cystine, suggests that the sparingly soluble glycoprotein is a giant macromolecule composed of at least two kinds of amino-acid containing structures associated through intermolecular - S - S - bonds. It is possible to conceive of the gel-like macromolecules as being built up from these two kinds of structures that differ only in their size, their content of total amino acid and in the frequency with which the acidic amino acid and cystine molecules occur in their amino acid-containing moiety and possibly their carbohydrate content and composition. One of the two amino-acid containing structures can be identified, after solubilization of the sparingly soluble material, as the typical water-soluble blood-group specific glycoprotein already well characterized and containing relatively small amounts of aspartic and glutamic acids and cystine. The second kind of structure m a y be similar to the first in overall structural pattern, but contains alarge proportion of total amino acids and especially of the acidic amino acids and cystine, or it may be composed of amino acids alone. An intermolecular - S - S - bonded association of these two types of molecule could give rise to a very large macromolecular structure that, because of its size, would most probably have low solubility and would presumably break up into its components when treated with simple reducing agents such as thioglycolate and sulphite. So far the second kind of structure has not been isolated as a homogeneous molecular species. All preparations obtained however have shown considerable group specific activity, which indicates that this second structure m a y contain some specific determinant carbohydrate groups and thus be glycoprotein in character, Only a few sparingly soluble glycoproteins have been examined, and these show certain differences in behaviour with reducing agents and in chemical composition. Until the appropriate data obtained from a larger number of preparations is available, the suggestions brought forward in this paper must be considered tentative. There is evidence that the overall size and conformation of the group specific glycoproteins influence the extent and firmness with which they combine with the corresponding antibody. I t is possible therefore that much useful information will be forthcoming to explain these and other observations if it is known more exactly how the very large and sparingly soluble glycoproteins differ from those that are of smaller molecular size and are readily soluble in water.
ACKNOWLEDGEMENTS
One of us (J.R.D.) thanks the Royal Society and the Nuffield Foundation for the award (1962-1963) of a Commonwealth Bursary. We thank Dr. A. PUSZTAI, Dr. J. M. CREETH and Dr. WINIFRED WATKINS for much useful discussion and advice. REFERENCES I 2 3 4 5
D. AMINOFF,W. T. J. MORGAN AND W. M. WATKINS,Biochem. J., 46 (195o) 426. R. A. GIBBONS, W. T. J. MORGAN AND M. N. GIBBONS, Biochem. J., 60 (1955) 428. A. PUSZTAI AND "~V. T. J. MORGAN, Biochem. J., 80 (1961) lO 7. W. T. J. MORGAN AND H. K. KING, Biochem. J., 37 (1943) 640. A. PUSZTAI AND W. T. J. MORGAN, Biochem. J., 80 (1961) lO 7.
Biochim. Biophys. Acla, IOI (1965) 3oo-314
314 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20
J. R. DUNSTONE, W. T. J. MORGAN
M. N. GIBBONS, Analyst, 80 (1955) 268. C. J. M. RONDLE AND W. T. J. MORGAN, Biochem. J., 61 (1955) 586. N. NELSON, J. Biol. Chem., 153 (1944) 375. G. E. FRANCIS,\¥. MULLIGAN AND A. ~VORMALL,Isotopic Tracers, The Athlone Press, London, ISt Ed., 1959, p. 277. E. J. CoNwAY, Microdiffusion Analysis and Volumetric Error, Crosby-Lockwood, London, 4th Ed., 1957. L. WARREN, J. Biol. Chem., 234 (1959) 1971. M. DUBOlS, K. A. GILLES, J. K. HAMILTON, ['. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956) 35 ° • M. P. TOMBS, F. SOUTER AND N. F. MACLAGEN,Biochem. J., 73 (1959) 167. \¥. T. J. MORGAN AND H. IK. KING. J., 37 (1943) 640. t2. A. GIBBONS AND W. T. J. MORGAN, Biochem. J., 57 (1954) 283. R. IR. RACE AND R. SANGER, Blood Groups in Man, Blackwell, Oxford, 4th Ed., 1962. S. MOORE, D. H. SPACKMAN AND "W. H. STEIN, Anal. Chem., 3 ° (1958) 1185. A. PUSZTAI AND x~V.T. J. MORGAN, Biochem..]., 88 (1963) 546. E. SCHRAM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. A. PUSZTAI AND \¥. T. J. MORGAN, Biochem. J., 93 (1964) 363.
Biochim. Biophys. Acta, i o i (1965) 3oo-314
BIOCHIMICAET BIOPHYSICAACTA
BBA 85065 F U R T H E R OBSERVATIONS ON T H E G L Y C O P R O T E I N S IN HUMAN OVARIAN CYST F L U I D S
J. R. DUNSTONE" AND Vq. T. J. MORGAN The Lister [nstit*tte of Preventive Medicine, London (Great Britain)
(Received August I6th, 1965)
SUMMARY An examination has been made of a number of ovarian cyst fluid glycoproteins that are sparingly soluble in water but are similar in general composition to the readily soluble blood-group specific glycoproteins obtained from the same source. Ultracentrifugal analysis of approx, o.i % w/v solutions of the sparingly soluble materials indicate that they have much larger sedimentation coefficients (s2o,w) and molecular weights than have the water-soluble glycoproteins. The sparingly soluble substances have more total amino acids and proportionally more aspartic and glutamic acids and cystine and less serine and threonine than have the soluble glycoproteins. The sparingly soluble substances mostly dissolve in buffered (pH 6.8-7.0, 0.05 M) solutions of thioglycolate, sulphite or cysteine, reagents that bring about the scission o f - S - S bonds. The behaviour and properties of the sparingly soluble glycoproteins suggest that they arise through the building up of relatively small glycoproteins to larger macromolecules through the formation o f - S - S - intermolecular associations. A number of ways whereby the formation of these sparingly soluble glycoproteins could occur are discussed.
INTRODUCTION Investigations on the chemical structure and immunological properties of human blood-group specific glycoproteins have mostly been made on the phenolinsoluble, water-soluble materials remaining after extraction of the freeze-dried contents of pseudomucinous ovarian cysts with cold 9o-95% w/w phenol 1-~. This method of preparation gives water-soluble group specific glycoproteins that have been well characterized. In most instances chemically similar glycoproteins that are sparingly soluble in water, and about which little is known, are also obtained. Digestion of the sparingly soluble material with pepsin at p H 2, or treatment b y ultrasonic irradiation, gives serologically active blood-group specific glycoproteins that are Present address: Department of Physical Biochemistry, The Australian National University, The John Curtin School of Medical Research, Canberra, A.C.T., Australia. Biochim. Biophys. Aela, ioi (1965) 3oo-314
OVARIANCYST GLYCOPROTEINS
301
readily soluble in water. However, with both methods of solubilization there is some uncertainty about the nature of the changes and extent of the degradation induced. It seems probable, however, that the glycoprotein that is sparingly soluble in water is a very large macromolecule that is easily changed by the cleavage of a few bonds into substances of smaller size which are readily soluble in water. The results of some preliminary attempts to obtain further information about the origin and nature of the sparingly soluble gel-like glycoproteins are given in the present paper. EXPERIMENTAL
Ovarian cyst glycoproteins. The glycoproteins are isolated from the freeze-dried contents of human pseudomucinous ovarian cyst adenoma by extraction at room temperature with 95 % w/w "liquid" phenol 4. The material remaining insoluble after thorough extraction with phenol is washed with a small amount of ethanol to remove excess phenol, suspended in water and dialyzed in Visking tubing. After dialysis is complete the viscous, gel-like, opalescent suspension is separated into water-insoluble and water-soluble components by centrifugation. The water-insoluble material is washed repeatedly with water and recovered by centrifugation at 30 ooo × g for 3 h. These glycoprotein preparations, which are mostly soluble to the extent of forming very dilute solutions (at most o. I °/o w/v) in water, are those used in the experiments described in this paper. The glycoprotein remaining in solution after dialysis of the phenol-insoluble preparation described above is the water-soluble specific blood-group substance. Sedimentation. Sedimentation-velocity experiments were performed in a Spinco Model E ultracentrifuge at 50 740 rev./min using the An-E rotor and sedimentation coefficients (expressed as Svedberg units: S) were corrected to water at 20 °. The runs were made at 25 ° and were controlled by the rotor temperature indication control unit. The partial specific volume of the substance was taken as 0.625, as reported by PUSZTAI AND ~V[ORGAN5. All measurements, except where stated otherwise, were made in 0.02 M KH2POa-K2HPO a buffer (pH 6.8) adjusted to 11 o.I with KC1. Chemical analyses. The preparations after drying from the frozen state were further dried over P205 for 30 min at 78° and 1-2 mm Hg pressure before analysis. The methods used for the determination of fucose 6 and for hexosamine 7 and reducing sugars s after acid hydrolysis were those used in earlier work. Nitrogen was determined by a micro-Kj eldahl procedure 9 followed by microdiffusion and titration in a standard Conway unit 1°. Sialic acid was determined by the method of WARREN11 after hydrolysis in o.I N H2SO4 for I h at 80 °. The carbohydrate content of solutions obtained directly from DEAE-cellulose columns was determined by the phenol-sulphuric method TM, while the amino acid content of these solutions was estimated by measuring the absorbance at 210 m/, (ref. 13). Serological activity. This was determined by the capacity of substances to inhibit in specific haemagglutination tests14,15. Glycoproteins possessing A, B, H and Le a activity le were investigated. Quanfitative amino acid analyses. These were carried out by the method of MOORE, SPACKMANAND STEIN17 as used by PUSZTAI AND •ORGAN is. In most experiments the glycoprotein was first oxidized with performic acid (16 h, --2 °) to convert cystine and/or cysteine to cysteic acid 19. Under these conditions certain amino acids Biochim. Biophys. Acta, IOI (1965) 3oo-314
302
j.
R. DUNSTONE, W. T. J. MORGAN
are extensively destroyed and quantitative amino acid analyses were therefore made on a few preparations before and after performic acid oxidation. The fully corrected 18 results for one preparation obtained from cyst fluid No. 485 are given in Table I. It is evident that decomposition of threonine, serine, leucine, phenylalanine and tyrosine occurred as a result of the performic acid oxidation. Corrections to compensate for the degradation of threonine and serine by performic acid have been applied to all analyses, but no corrections have been made for the losses of the other amino acids. Some typical analytical results are given in Table II. TABLE I EFFECT
OF PERFORMIC
ACID OXIDATION
ON T H E A M I N O A C I D C O M P O S I T I O N
OF BLOOD-GROUP
SPECIFIC
SUBSTANCES
Oxidized a n d n o n - o x i d i z e d s a m p l e s of m a t e r i a l s p a r i n g l y soluble in w a t e r from c y s t fluid No. 485 were h y d r o l y z e d w i t h 6 N HC1 a t 114 i i 7 ° for 24 h a n d a n a l y z e d q u a n t i t a t i v e l y for t h e i r cons t i t u e n t a m i n o acids. The r e s u l t s are e x p r e s s e d as t h e m e a n v a l u e s of t r i p l i c a t e d e t e r m i n a t i o n s a n d are recorded as g a m i n o a c i d / i o o g of s u b s t a n c e , a n d h a v e been c o r r e c t e d for d e g r a d a t i o n d u r i n g acid h y d r o l y s i s is.
A m i n o acid
Without oxidation 1.74 5-95 3.18 2.33 2.33 0.97 i .39 1.o6 1.51 o.8o
With oxidation
A s p a r t i c acid Threonine Serine G l u t a m i c acid Pr oline Glycin e Alanine C y s t i n e (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine
1.74 5.43 2.97 2.27 2.33 I .oi i .44 1.o 3 1.46 o.77
i ,2o
i .0 5
o. 73 0.78 --
o. i o o. I I
T o t a l a m i n o acids
23.97
21.88
o, 17
Complete amino analyses were not always carried out, as it appeared from earlier results 18 that sufficient data to allow the characterization of the amino acid moiety of blood-group specific glycoproteins could be obtained without the determination of the basic amino acids, lysine, histidine and arginine, which together make up less than IO% of the total amino acids. Column chromatography on DEA E-cellulose. Chromatography of dilute solutions of the glyeoproteins was carried out on ~5o cm × 2 cm columns of DEAE-cellulose, as already described ~°. The substance was eluted with NaC1 solution (continuous NaC1 gradient, usually o.oi M - I M), but in some experiments other eluants followed the NaC1 gradient in order to recover as much material as possible from the column. Unless otherwise stated 5o-ml fractions were collected at a rate of 50 ml/h and the elution patterns were determined by analyzing I-ml portions from each fraction. Solubilization experiments. The phosphate buffer (o.oi M KHo,PO a, o.oi M Biochim. t~iophys. Acla, i o i (1965) 3 o o - 3 i 4
303
O V A R I A N CYST G L Y C O P R O T E I N S TABLE
II
THE AMINO ACID COMPOSITION OF SOME SPARINGLY SOLUBLE AND THE CORRESPONDING WATER*SOLUBLE GLYCOPROTEINS FROM THE SAME OVARIAN CYST FLUID E x p e r i m e n t a l d e t a i l s a r e g i v e n in t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d a s (a) g o f a m i n o a c i d / i o o g o f s u b s t a n c e ; ( b ) / * m o l e s o f a m i n o a e i d / i o o / , m o l e s o f t o t a l a c i d s e s t i m a t e d . A n a l y t i c a l v a l u e s f o r m a t e r i a l s (i) s o l u b l e a n d (ii) s p a r i n g l y s o l u b l e i n w a t e r a r e g i v e n .
Amino acid Aspartic acid Threonine Serine Glutamicaeid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
A substance No. 485
B substance No. 413
H substance No. 476
Le a substance No. 35 °
(i)**
(i)*,'**
(i)*,*'"
(i)*'.***
(a) 0-75 5-19 3.03 1.16 1.74 0.6o 0.96 0.45 0.86 0.60 0.59 ---
15.93
(ii)
(ii)
(ii)
(ii)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
4 .0 31.2 20.6 5 .6 lO.8 5.7 7-7 2.7 5.2 3.3 3 .2 ----
7-3 21.6 15. 9 8.3 II.I 7.1 8.8 4.4 6. 3 3.3 4.5 0. 3 0. 5 0.6
0.79 3.80 1.98 0.73 2.00 0.58 1.39 o.ii 0.68 o.31 0.34 o.15 0.28 --
5 , i 1.82 27.4 3.97 16.1 2.44 4.3 2.11 14. 9 1.98 6.6 0.93 13. 4 1.14 0,8 0.96 5 .0 1.38 2.o o.91 2.2 1.35 0.8 - 1. 4 -o.31
8.2 2o.1 14.o 8.6 lO. 4 7.5 7.7 4.8 7 .1 4.2 6.2 --1.2
0.70 3.94 2.21 o.91 1.6o °.51 1.14 o.15 0.59 0.30 0.24 o.io 0.20 --
4.4 3.3 ° 31.6 4.65 19. 3 3.35 5.4 4 .26 12.1 2.58 5.9 1.23 i i . o 2.31 i . o 1.52 4-3 2.43 1.9 1.24 1.6 2.83 0. 5 0. 5 i . o 0.32 -0.55
9.5 15.o 12.2 ii.i 8.6 6.3 io.o 4.8 8.0 3.6 8. 3 0. 5 0. 7 1. 4
o.4o 3.66 1.69 0.59 1.66 0.55 1.o 5 o.ii 0.69 o.31 0.69 0.05 0.27 --
2.9 2.85 29.8 4.07 15.6 2.41 3.8 3.56 13.9 1.94 7.0 1.21 11. 3 2.00 0.9 1.14 5-7 1.88 2.2 0.80 5.1 2.20 0. 3 0.08 1. 5 - -o.15
13.14
19.3 °
12.59
3o.82
11.72
24.29
1.78 4.74 3.07 2-24 2.34 0.97 1-43 0.98 1.36 0.80 1.o9 0.09 o.15 o.17 21.11
* S o l u b l e in s a t u r a t e d ( N H a ) 2 S O a a t 6o °. ** I n s o l u b l e in s a t u r a t e d (iXTH4)2SO4 a t 6o °. "** C a l c u l a t e d f r o m d a t a o f PUSZTAI AND MORGAN 18.
K2HPO 4, o.o6 ~ KC1, p H 6.8) used in the solubilization experiments contained o.o5 M reducing agent, cysteine or sodium thioglycolate. The sulphite buffer was 0.048 M Na2HPO 4, 0.02 M KH2PO 4, 0.03 IM Na2SO 3, o.o13 M NaHSO z (pH 7.0). RESULTS
An examination of the solubility of the sparingly soluble glycoprotein preparations indicated that solutions of m a x i m u m concentration of about o.I °/o w/v could be prepared in most instances b y dispersing the geMike materials (about o.5% w/v) (a) in anhydrous formamide, followed by dialysis of the suspension against distilled water or buffer solutions at 2 ° and centrifuging at 30 ooo × g, I h or (b) in distilled water by stirring vigorously for up to 3 h and then removing undissolved substance by centrifuging (30 ooo × g, 3 h). The fact that small amounts of material continue to dissolve after repeated extraction of the centrifuge deposits with water indicates that the preparations have definite, but limited, solubility.
Ultracentrifugal examination Solutions were prepared, as described above, from sparingly soluble glycoprotein preparations obtained from cyst fluids No. 485 (group A) and No. 413 (group B). The ultracentrifugal patterns obtained with dilute solutions of both materials showed Biochim. Biophys. Acta,
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(b) lO.3 16.4 ii.o 11.6 8.1 7.7 lO.8 4-5 7.7 2.9 8.1 0.2 -0.5
304
j. R. DUNSTONE, W. T. J. MORGAN
Fig. i. S e d i m e n t a t i o n d i a g r a m s of solutions p r e p a r e d from t h e s p a r i n g l y soluble glycoproteins in t h e presence, or absence, of thioglycolate. All p h o t o g r a p h s were t a k e n 24 m i n after r e a c h i n g a speed of 5 ° 74 ° r e v . / m i n ,The p h a s e plate angles were (a), (b) a n d (d), 45°; (e) a n d (f), 4o°; (c) 55°; (g) 6o °. D i a g r a m s (a), (b), (c) a n d (d) were o b t a i n e d w i t h m a t e r i a l s f r o m o v a r i a n cyst No. 485 . D i a g r a m s (e), (f) a n d (g) with m a t e r i a l s f r o m o v a r i a n c y s t No. 413 . T h e m e t h o d s of p r e p a r a t i o n of t h e v a r i o u s s a m p l e s are given in t h e text.
more than one sedimenting boundary (Fig. I a and e). The boundaries were not sharp, most probably owing to the low concentration and the relatively high degree of polydispersity. Account could not be taken of the Johnston-Ogston effect, nor of the effect of the concentration dependence of the sedimentation coefficients. The s%0,w values of the fastest sedimenting components were greater than 15 S and this considerably higher than those most frequently found (S°2o,w lO-12 S) for the upper molecular range of values determined for the soluble blood-group specific substances. This indicated that in these preparations were molecules with sizes much larger than those normally encountered in the water-soluble glycoproteins.
Chemical composition The qualitative and quantitative carbohydrate composition of the two waterinsoluble substances studied in the ultracentrifuge (Tables I I I and IV) indicated that in each instance the materials have similar compositions to those of the water-soluble glycoprotein obtained from the corresponding cyst fluid. Amino acid analyses revealed that each of a number of sparingly soluble glycoproteins had a greater amount of total amino acid than did the corresponding water-soluble glycoproteins and they contained a higher proportion of aspartic acid, glutamic acid and cystine and lower proportions of threonine, serine and proline (Table II). The presence of a greater proportion of cystine in the sparingly soluble preparations was confirmed after oxidizing each material with performic acid. A peak corresponding to that of cysteic acid replaced that of cystine and the amount of cysteic acid was found to be equivalent to that of the cystine previously found. The occurrence of relatively large amounts of cystine in the sparingly soluble materials suggested that their low solubility might be attributed to the formation Biochim. Biophys. Acla, IOX (1965) 3oo-314
O V A R I A N CYST G L Y C O P R O T E I N S TABLE
305
III
T H E COMPOSITION OF FRACTIONS O B T A I N E D FROM T H E S P A R I N G L Y SOLUBLE P R E P A R A T I O N FROM O V A R I A N C Y S T NO. 4 8 5 ( G R O U P A ) A F T E R R E D U C T I O N W I T H T H I O G L Y C O L A T E A N D C H R O M A T O G R A P H Y ON D E A E - c E L L U L O S E
E x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d a s (a) g o f a m i n o a c i d / i o o g o f s u b s t a n c e ; (b) p m o l e s o f a m i n o a c i d / i o o p m o l e s of the amino acids estimated. Other values a r e e x p r e s s e d a s g / i o o g o f s u b s t a n c e . F r a c t i o n s a s s h o w n in F i g . 2.
Original sz~bslance
W e i g h t (rag) Nitrogen Fucose Hexosamine Reducing sugar Sialic acid S e r o l o g i c a l a c t i v i t y (~,g)
272 6.3 15 25 45 3 .8 --
(a) Asparticacid Threonine Serine Glutamicacid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
Fraction numbers
1.78 4-74 3.07 2.24 2.34 0.97 1.43 o.98 1.36 0.80 1.°9 0.09 o.15 o.17 2 i. i i
I
2
3
4
5
43 5.5 16 27 58 3.4 0.2
85 5.6 15 25 51 4 0.25
19 5.7 iI 25 46 5.1 0.6
iI 4.2 7 16 48 2.7 0. 7
31 5.6 15 23 5i 3.6 0.2
(~) (a)
(b) (a)
(b) (a)
(b) (a)
(b) (a)
7.3 21.6 15.9 8. 3 ii.i 7.1 8.8 4.4 6.3 3.3 4-5 0. 3 0. 5 o.6
3-9 33 .0 21.8 5.1 8.9 6.7 8.9 2.0 4.7 2.5 1-9
1.17 4-4 ° 2.98 1.53 1.5o 0.85 1.12 o.71 1.o4 0.57 0.67
o.17
7.5 16.8 14.8 lO. 5 7.8 9.9 8.4 5-4 7.4 2.9 4.3 1.8 0.9 1.6
l°.3 15. 5 13.9 9.9 6. 5 13.2 9.7 5-9 6.2 3.8 3-9 --
o.6
5.9 24. 9 18.8 7.0 8.8 9.1 8.5 3.9 6.o 2.9 3.4 --o.8
o-75 5.74 3.35 1.io 1.49 0.74 1.15 o.36 o.80 0-47 o.37 --o.13 16-44
16.66
1.86 3.72 2.89 2.88 1.66 1.38 1.39 1.32 1.62 o.71 1.o5 0.60 0.28 o.43 21.69
2.o7 2.78 2.20 2.18 1.13 1.49 1.3o 1.o7 I.iO 0.75 0.78 ---o.27 17-12
1.2
1.84 4.24 2.5 ° 1.94 1.45 i.oi 1.4 ° 0.62 1.11 0.57 0.60 o.16 ---
(b) 9.1 23.3 15.6 8.7 8.2 8.8 lO.3 3.4 6.2 2.8 3.0 o.6 ---
17,44
of intermolecular disulphide bonds and through them the formation of macromolecular complexes of much larger size. To test this suggestion the solubility behaviour of some sparingly soluble glycoproteins was examined in dilute, neutral solutions of reducing agents, such as thioglycolate, cysteine and sulphite, that are known to bring about the scission of disulphide linkages. Preliminary experiments indicated that the glycoproteins sparingly soluble in water obtained from cyst fluids No. 485 (A) and 413 (B) dissolved readily in a phosphate-thioglycolate or phosphate-cysteine buffer at pH 6.8. The glycoprotein preparations obtained from cyst fluids No. 476 (H) and 35o (Lea) however were less readily soluble under similar conditions. Substance No. 476 dissolved to the extent of about 5o% in 2o h and dissolved almost completely after a longer time. After several weeks the reducing agents dissolved not more than about 2o% of substance No. 35o and thereafter the solubility remained unchanged. Solution of the substances in phosphate-sulphite buffer (pH 7.o) was more rapid, the glycoprotein materials No. 485 and 413 dissolving immediately and No. 476 in about Biochim. Biophys. Acla, i o i (1965) 3 o o - 3 1 4
306
j.R.
D U N S T O N E , W. T. J. MORGAN
TABLE IV THE COMPOSITION OF FRACTIONS OBTAINED FROM THE SPARINGLY SOLUBLE PREPARATION FROM OVARIAN CYST NO. 413 (GROUP B) AFTER REDUCTION WITH THIOGLYCOLATE AND CHROMATOGRAPHY O N D E A E - C E L L U L O S E E x p e r i m e n t a l d e t a i l s a r e g i v e n in t h e t e x t . A m i n o a c i d s a r e e x p r e s s e d as (a) g o f a m i n o a c i d / I o o g of s u b s t a n c e ; (b)/~moles of a m i n o a c i d / i o o / , m o l e s of the a m i n o acids e s t i m a t e d . O t h e r values are e x p r e s s e d as g / i o o g o f s u b s t a n c e . F r a c t i o n s as s h o w n in Fig. 3.
Original substance
Fraction numbers I
W e i g h t (rag) N Fucose Hexosanline Reducing sugar Sialic a c i d S e r o l o g i c a l a c t i v i t y (pg)
Aspartic acid Threonine Serine Glutamieacid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Tyrosine Phenylalanine Methionine Total amino acids
300 5,9 13 25 49 6. i --
6 3.2 16 21 42 I. i 0.07
(a)
(b)
(a)
1.82 3.97 2.44 2.11 1.98 0.93 1.14 0.96 1.38 o.91 1.35 --
8.2 2o.1 14-o 8.6 lO. 4 7.5 7.7 4.8 7 -1 4.2 6.2
0.90 2.70 1.28 0.60 1.o 7 0.64 0.73 0.07 0.30 0.27 0.20
o.31
1.2
19.3 °
2
-8.76
3
20 4-3 17 21 45 1.7 0.08
(b) (a)
80 4.7 12 22 41 5-7 0.09
5
9 5 -1 ii N.D.* N.D. 5 .8 0. 4
(a)
(b)
1.49 3.56 2.52 1.72 1.37 0.87 1.24 0.63 0.88 0.53 0.78 0.25
8.1 1.53 21.6 3.32 17.3 2.57 8.5 1.77 8.6 1.6o 8.4 1.o4 i o . i 1.26 3.8 0.70 5.4 1 . ° 9 2.9 0.43 4.3 0.76 i . o o.17 -0.09 0.22
8.1 1.81 19.2 4.33 8.3 2.74 8. 3 2.38 9.6 1.66 9.5 1.26 9.8 1.44 4.0 o,66 6. 4 1,6o 2.3 o,78 4 .0 1,3o 0.6 - 0. 4 - i . o 1.3o
16.55
21.26
1,54 3,58 2,42 1,27 i,ii o.71 1-o9 0.26 0.69 0.35 0.37
9.6 25. 7 I9.1 7.2 8.0 7 .8 lO.2 1.8 4.9 2.2 2.3
--
0.22
1.2
-15.84
(a)
(b)
57 5 .8 9 21 37 5.5 0.2
(b)
8.7 28. 7 15.6 5.2 11. 9 lO.9 IO.5 o. 7 3-3 2.6 1.9
13.61
4
(a)
(b) 7.4 19-9 14.2 8.8 7.9 9.2 8.8 3.0 7.5 3.2 5.4 --4.7
" N.D. = Not determined.
36 h. Glycoprotein preparation No. 35o dissolved completely only after repeated treatment with freshly prepared sulphite. The ease with which preparations No. 485 and 413 dissolved in the reducing agents suggested that their behaviour in buffered o.o5 M thioglycolate (pH 6.8) should be examined more closely. Portions of the substances were therefore dispersed (about o.5% w/v) in anhydrous formamide as described earlier and solutions of the soluble fractions in water recovered. Portions of these solutions (approx. o.1% w/v) from each of the glycoproteins were dialysed against (a) phosphate buffer and (b) phosphate -thioglycolate buffer. The dialysed solutions were then examined in the ultracentrifuge. The fastest sedimenting boundary in the non-reduced substance No. 485 ( F i g . I a ) disappeared after reduction (Fig. Ib). Longer centrifuging caused the latter peak to separate into two components with S%o,w values of about IO S and 8.5 S. Similar changes were produced under the same conditions by the reduction of a dilute Biochim. Biophys. Acta,
i o i (1965) 3 o o - 3 1 4
307
OVARIAN CYST GLYCOPROTEINS
solution of glycoprotein No. 413, but they were less marked. Nevertheless differences in the relative proportions of the components after reduction were observed: Fig. ie, f. Attempts to reverse the dissociation were made by removing the reducing agent from preparation No. 485 by dialysis against oxygenated water at 2 ° for several days. No changes in the ultracentrifuge pattern were produced by this treatment (Fig. Id), suggesting that none of the original and heavier material was reformed. Solutions prepared by dissolving the sparingly soluble glycoproteins in thioglycolate buffer without previous dispersion in formamide, were similarly investigated. More of the glycoproteins (No. 485 and 413) dissolved than after treatment with formamide and the ultracentrifugal patterns obtained indicated that the fastest moving components (Fig. Ia and e) before reduction were not detected in the reduced preparations (Fig. ic and g). Longer centrifuging of preparation No. 485 (Fig. IC) showed it to be composed of two major components (s%0,w values about IO S and 8 S), but the material from preparation No. 413 sedimented as a single peak with the characteristics of a highly polydisperse material. The peak areas, when corrected for sectorial dilution, decreased in value as the run progresssed, indicating the probable continuous sedimentation of very large macromolecules. B e h a v i o u r on D E A E - c e l l u l o s e columns
The behaviour of the solubilized glycoproteins on columns of DEAE-cellulose was examined. The sparingly soluble glycoproteins, about 300 mg of each, were dis1.2F 1 4 - - 1 - - 4 ~ r - - ~ . - 2 ~ 3
~:~
4
/
!
1.0-
0.8
0.6 u £1
1.0
m
< 0.4
0.8
0.2
3.4~
Z
0.2 ~. 0
20
40 Tube number
60
80
100
Fig. 2. C h r o m a t o g r a p h y on D E A E - c e l l u l o s e of s o l u t i o n s of s p a r i n g l y soluble g l y c o p r o t e i n o b t a i n e d f r o m o v a r i a n c y s t fluid No. 485 (group A). D e t a i l s of t h e p r e p a r a t i o n are g i v e n in t h e t e x t . Cont i n u o u s g r a d i e n t e l u t i o n w i t h NaC1 is s h o w n b y t h e line . . . . . . C o n t e n t s of tubes, each c o n t a i n i n g 50 m l were pooled as s h o w n b e t w e e n a r r o w s to give t h e v a r i o u s fractions. 0 - - - 0 , a b s o r b a n c e a t 21o m ~ ; Q)--C), a b s o r b a n c e a t 485 m/z ( p h e n o i - s u l p h u r i c a c i d method13).
Biochim. Biophys. Acta, i o i (1965) 3oo-314
308
j. R. DUNSTONE, W. T. J. MORGAN 14-1 ~
2
~4~
3
~
4
1.2
1.Q
0.8
0.6
1.0 ou} ~" 0.4
/
-0.8
/
// /
0.2
J
-o.6~
/ -0,4~
0.2 O
0
2o
40
6o
U
80
Tube number"
Fig. 3. C h r o m a t o g r a p h y on DEAE-cellulose of solutions prepared from the sparingly soluble p r e p a r a t i o n from ovarian cyst fluid No. 413 (group B). Solutions, obtained by treating 3oo m g portions of material with (a) thioglycolate and with (b) water, as described in the text, were applied to the column. Continuous gradient elution with NaC1 is s h o w n by the line . . . . . . Contents of tubes, each containing 5o ml, were pooled as s h o w n between the arrows to give the various fractions. Absorbance at 485 mff (phenol-sulphuric acid m e t h o d TM) for (a) O - - Q and (b) O - - O .
solved in thioglycolate-containing buffer (5o ml, p H 6.8), dialysed against distilled water to give about o.5% w/v solutions which were then made o.oi N with respect to NaC1 and applied to a DEAE-cellulose column (15o cm × 2 cm). The elution patterns obtained for preparations No. 485 and 413 are given in Figs. 2 and 3, respectively. The values plotted in Fig. 2 are based on carbohydrate determination by the phenol-sulphuric acid method 12 and absorbance at 21o m# (ref. 13). The curves obtained for substance No. 485 (Fig. 2) indicated that there were no appreciable amounts of a substance composed only of amino acids. A similar result was obtained for substance No. 413 . Preparation No. 485 was resolved into several fractions. The first four were obtained by gradient elution with NaC1, the last (Fraction 5) b y elution with o.2 M NaOH; this fraction emerged with the NaOH front. Analytical values obtained for these fractions are given in Table I I I . A small amount of material was eluted from the column when phosphate buffer (0.2 3/[ NaH~PO4-Na~HPO4, pH 6.8) was used as eluant immediately after the NaC1 gradient was complete and before elution with NaOH started, but because the fraction was not free from buffer ions, the analytical figures obtained are not included in Table I n . However, the following values obtained for the material eluted with phosphate buffer: aspartic acid, 12.o; threonine, 12.o; serine, 12.2; glutamic acid, 11.7; proline, 5.7; glycine, 15.4; alanine, 8.5; cystine Biochim. Biophys. Acta, IOI (1965) 3oo-314
OVARIAN CYST GLYCOPROTEINS
309
(half), 6. 7 (expressed as/,moles of amino acid/ioo/,moles total amino acids estimated), are of considerable interest when compared with the amino acid distribution normally obtained (Tables II, III) and especially when compared with that for the glycoprotein recovered in the last fraction to be eluted with NaC1 (Fraction 4). Fraction I (Fig. 2, Table III) closely resembled in chemical composition the water-soluble material obtained from the same cyst fluid (Table II). Fractions 2, 3 and 4 (Fig. 2, Table III) and the material eluted with phosphate buffer contained progressively larger proportions of aspartic acid, glutamic acid, cystine (half) and glycine and smaller proportions of threonine, serine and proline. Fraction 5 (Table III) resembled more closely in composition the original material. Distinct variations in the small amounts of sialic acid in the various fractions were also observed, and most probably influenced to some extent the elution behaviour of the fractions collected. The total amount of amino acid in each fraction was, with the exception of Fraction 3, Table III, smaller than the amino acid content of the original preparation. Fractions I and 2 (Table III) had similar ultracentrifugal patterns characterized by single "hypersharp" peaks with only a very small amount of faster sedimenting material and compared closely with those obtained for the water-soluble glycoprotein isolated from the same cyst fluid. The other fractions were considerably more heterogeneous and Fraction 5 contained two major components. All fractions were active in haemagglutination inhibition tests; Fractions I and 2 (Table III) were as active as the highly-purified, water-soluble preparation (Table II) from the same cyst fluid. The other fractions were less active. The glycoprotein No. 413 was similarly separated into four fractions by gradient NaC1 elution from a DEAE-cellulose column (Fig. 3). Again a last and fifth fraction was recovered by elution with 0.2 M NaOH. The analytical data for these fractions are given in Table IV. Each fraction contained a greater proportion of aspartic acid and glutamic acid (Table IV) than did the corresponding water-soluble material (Table II). The proportions of threonine and serine in Fractions I and 2 were similar to those of the water-soluble material; the other fractions contain somewhat less threonine. The proportion of cystine (half) was increased in the later fractions (Table IV, Fractions 3, 4 and 5). The first fraction, which contained the smallest amount of total amino acids, resembled more closely in Fattern the water-soluble preparation than did any of the other fractions. Fractions 3 and 5 were examined in the ultracentrifuge. The sedimentation diagrams indicated in each instance one maj or component with smaller but nevertheless significant amount of slower sedimenting material. All fractions were serologically active ; Fractions, I, 2 and 3 were as active, within the limits of the tests, as was the purified water-soluble blood-group substance obtained from the same cyst; Fractions 4 and 5 were less active. In order to make a valid interpretation of these results it was necessary to establish that the fractions obtained from the DEAE-cellulose columns were the materials solubilized by reduction of the sparingly soluble glycoprotein with thioglycolate and not soluble components already present in the interstices of the gel-like preparations. Portions (300 mg) of sparingly soluble substance No. 413 were suspended in distilled water (5o ml) and stirred vigorously for several days. The suspensions were then centrifuged (30 ooo × g, 3 h) and the supernatant solutions, suitably concentrated, were chromatographed on DEAE-cellulose columns. The total amount of soluble material was small compared with that obtained from the same glycoBiochim. Biophys. dcta, i o i (1965) 3oo-314
31o
j.R.
DUNSTONE, W. T. J. MORGAN
protein preparation after treatment with thioglycolate buffer. The elution patterns obtained for the material soluble in water and in thioglycolate buffer are given in Fig. 3 and indicate that the soluble substances recovered after the thioglycolate treatment could not be obtained by simple extraction with water and thus, were not soluble glycoproteins trapped in the interstices of the gel structure. The ready solubility of the sparingly soluble glycoprotein in reagents which specifically reduce disulphide bonds prompted an attempt to determine the amount of free sulphydryl groups in one preparation, after reduction with sulphite using p-chloromercuribenzoate. The sparingly soluble glycoprotein (IOO rag) obtained from cyst fluid No. 485 was suspended in 5.0 ml ofo.2 M acetate buffer (pH 5.3) containing 0.05 M Na2SO 3. After the material had dissolved 3.0 ml of 5 mM sodium p-chloromercuribenzoate was added and 16 h later a portion of the solution was titrated with 5 mM cysteine, using sodium nitroprusside as external indicator to determine the amount of p-chloromercuribenzoate unreacted with the free sulphydryl groups of the reduced glycoprotein. The glycoprotein contained 8.1/,moles of sulphur, calculated from the amount of cystine present (Table II). The results of the experiment showed that 3.6/*moles of sulphydryl sulphur were present after reduction. The scission of a disulphide bond with sulphite proceeds according to the equation: R-S-S-R
+ SOa '~- = R - S - + R S - S - O 3-
if all the sulphur in the original glycoprotein were present as disulphide sulphur, 4.05/,moles of sulphydryl sulphur should have been obtained; if incomplete reduction had occurred the value would be lower. On the other hand, if some of the sulphur of the original glycoprotein were present as sulphydryl sulphur the yield should be greater than 4.05/*moles. Therefore, it would appear that most of the sulphur in the glycoprotein is present as disulphide sulphur. An attempt to determine the free sulphydryl groups in the sparingly soluble preparation without previous reduction was unsuccessful because of the presence of the thick opalescent gel during this determination. Thus,
Digestion with pepsin The composition of the water-soluble glycoproteins obtained from the sparingly soluble gel-like preparation after digestion with pepsin was determined. Glycoproteins (300 nag) No. 485 and 413 were completely solubilized as described in Table V. The analytical figures for the substances before treatment with pepsin are given in Table I I. The first and largest fraction from each solubilized glycoprotein, at a concentration of about I °/o (w/v) in water, precipitated within the limits of 48 and 52% (v/v) ethanol, and in this property resembled closely the water-soluble blood-group substances. The analytical data for these fractions, obtained from glycoproteins No. 485 and No. 413 were N, 5.8 and 5.1; fucose, 17 and 14; total amino sugar, 27 and 25; total reducing sugars, 55 and 54, and sialic acid, 3.1 and 4.6, respectively. The amino acid composition in each instance (Table V) was closely similar to that of the corresponding water-soluble blood-group substance given in Table II except for a somewhat greater proportion of cystine (half). On examination in the ultracentrifuge both substances sedimented as single peaks, but were somewhat more polydisperse than the corresponding water-soluble glycoproteins. The smaller fractions precipitating at higher ethanol concentrations (Table V) Biochim. Biophys. Acta, i o i (1965) 3oo-314
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TABLE V CHEMICAL PROTEINS
COMPOSITION SPARINGLY
OF
THE
SOLUBLE
IN
WATER
SOLUBLE
WATER
AFTER
GLYCOPROTEINS TREATMENT
WITH
ISOLATED
FROM
THE
GLYCO-
PEPSIN
Portions of the sparingly soluble preparations (300 mg) were suspended in o.i M citric acidNaoHPO 4 buffer (pH 2.2) and incubated with crystalline ( x 2) pepsin (2 mg) at 37 ° for 16 h. The resulting solutions were dialysed and the glycoproteins isolated by repeated fractionation with ethanol. The materials were then dissolved in water, centrifuged at 3° ooo > g for I h and the s u p e r n a t a n t solutions dried from the frozen state. Amino acids are expressed as (a) g of amino acid/ioo g of substance and (b) /~moles of amino acid/ioo #moles of total amino acids estimated. A substance No. 485 Weight (mg) Ethanol fraction (v/v%)
z3o 48-52 (a)
Asparticacid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Iso-leucine Leucine Methionine Total amino acids
23 52-59 (b)
(a)
39 59-75 (b)
0.86 4-5 1.38 5-7 5.8o 28.6 5.15 24.0 3.25 21.7 3.48 16.2 1.23 5.8 1.84 6.9 1.82 i i . i 2.48 11. 9 0.75 6.9 1.o7 8.0 1.o 3 8.0 1.53 9.5 0.64 3-7 1-o3 4.8 0.88 5.2 1.3o 6.1 o.41 2.2 o.61 2.6 0.49 2.3 0.78 3.3 -- - 0.27 I.O 17.16
2o.92
B substance No. 4z3
(a)
14o 48-52 (b)
(a)
32 52-59 (b)
(a)
2.09 8.2 1.o 3 5-9 2.o6 3.91 17.1 4.42 28.4 4-3 ° 2.84 14.1 2.29 16. 7 3.32 2.35 8.3 1.22 6. 4 2.81 2.53 11. 5 1.63 lO.8 2.80 1.26 8.8 0.74 7-5 1.63 1.37 8.0 1.17 io.o 1.59 1.82 7-9 0-47 3 .0 1.36 1.71 7 .6 0.87 5.7 1.41 0.79 3.1 0.48 2.8 0.64 1.36 5.4 0.48 2.8 0.97 0.64 22.o3
14.8o
23.53
3° 59-75 (b)
(a)
(b)
7.6 17.6 15. 5 9.3 11.9 lO-7 8.8 5.5 5.9 2. 4 3.6 1.2
2.88 5.59 2.64 4 .06 2.54 1-46 1.63 1.73 1.78 0.95 1.53 --
9.4 20-4 ii.o 12.o 9.6 8.5 8.0 6.2 6-7 3.1 5.1 --
26.79
s h o w e d g r e a t e r p r o p o r t i o n s o f c y s t i n e (half), a s p a r t i c a c i d a n d g l u t a m i c a c i d a n d l o w e r proportions of threonine and serine than did the first fractions and the corresponding w a t e r - s o l u b l e specific g l y c o p r o t e i n s ( T a b l e I I ) . T h e m a t e r i a l r e c o v e r e d f r o m t h e p e p s i n digests accounted for about two-thirds of the sparingly soluble material digested. DISCUSSION T h e g l y c o p r o t e i n s o c c u r r i n g i n o v a r i a n c y s t fluids t h a t h a v e b e e n m o s t t h o roughly examined have serological properties associated with blood-group specificity, are readily soluble in water and have molecular weights between 2-105 and 2.106 . I t is f r e q u e n t l y f o u n d h o w e v e r t h a t p a r t o f t h e v i s c o u s o p a l e s c e n t g l y c o p r o t e i n p r e p a rations obtained are sparingly soluble in water. These sparingly-soluble, gel-like materials resemble, in general chemical pattern and in certain physical properties, the soluble glycoproteins and can be readily solubilized with little change in composition by enzymic digestion or ultrasonic irradiation. The results now reported illustrate certain additional features that distinguish more clearly the sparingly soluble glycoproteins from the corresponding water-soluble p r e p a r a t i o n s o b t a i n e d f r o m t h e s a m e c y s t fluid. C h e m i c a l a n a l y s i s c o n f i r m s t h a t t h e s e substances are glycoproteins and their ultracentrifugal examination in dilute solution Biochim. Biophys. Acta, IOi (1965) 3oo-314
312
J.R. D U N S T O N E , W . T. j. M O R G A N
indicates that they have very much larger sedimentation coefficients than do the corresponding water-soluble substances. The results of more exact amino acid analyses also show that for the four sparingly soluble preparations examined, each has an amino acid pattern that is different in certain important respects from that of the corresponding water-soluble glycoprotein. The sparingly soluble preparations have an appreciably greater amount of total amino acid, a smaller proportion of threonine, serine and proline and a larger proportion of aspartic acid, glutamie acid and cystine than do the corresponding water-soluble glycoproteins. The properties of low solubility in water or buffers, of large molecular size, and of having enhanced proportions of cystine, suggest that the sparingly soluble glycoproteins are very large macromolecular species built up through the formation of a number of intermolecular disulphide (-S-S-) bonds. The demonstration that these sparingly soluble substances dissolve at neutral pH in simple reducing agents, such as thioglycolate and cysteine that are known to bring about cleavage of disulphide bonds, and that there is a simple relationship between the number of free - S H groups after sulphite reduction, and the total sulphur content, supports this suggestion. Some further evidence for this idea was obtained from ultracentrifugal analysis. The fastest sedimenting component in the original sparingly soluble preparations was absent, or considerably reduced in amount, when dilute solutions of these materials were treated with thioglycolate. The first and readily soluble fraction recovered from the sparingly soluble glycoprotein obtained from cyst No. 485, after solubilization in thioglycolate and separation on a DEAE-cellulose column, had an overall composition not unlike that of the water-soluble glycoprotein obtained from the same cyst fluid. Later fractions eluted with increasing salt concentration, however, had considerably more aspartic acid, glutamic acid and cystine than did the first fractions. The sparingly soluble glycoprotein obtained from cyst No. 413 after solubilization with thioglycolate and separation on a DEAE-cellulose column, also gave soluble components in which the proportions of threonine, serine and cystine were not dissimilar to those obtained for the water-soluble glycoprotein recovered from the same cyst fluid, but the early fractions contained a significantly higher proportion of aspartic and glutamic acids than did the glycoprotein originally soluble in water and obtained from the same cyst fluid. It is known however that homogeneous glycoprotein preparations are not necessarily obtained as a result of a single chromatography on DEAE-cellulose, as used in these experiments, and it is possible that the early fractions from the DEAE-cellulose column, while containing a relatively high proportion of glycoprotein similar in chemical composition to the water-soluble blood-group substances, remain contaminated with material that occurs more plentifully in the later fractions that are rich in acidic amino acids. The possibility of bringing about a more complete resolution by rechromatography was not investigated. The major water-soluble glycoprotein fractions obtained after digesting the sparingly soluble preparations with pepsin resembled the water-soluble blood-group substances in general chemical composition, but here again there was a significantly greater proportion of acidic amino acids and cystine in the solubilized product than in the corresponding soluble glycoprotein from the same cyst fluid. The fractions obtained by precipitating the digest with higher concentrations of ethanol however contained proportionally more acidic amino acids and cystine. Biochim. l~iophys. Acta, i o i ( i 9 6 5 ) 3 o o - 3 1 4
OVARIAN CYST GLYCOPROTEINS
313
The fact that a considerable proportion of the sparingly soluble glycoprotein after solubilization with thioglycolate or sulphite can be recovered as a readily soluble glycoprotein that is relatively poor in aspartic and glutamic acids and cystine, suggests that the sparingly soluble glycoprotein is a giant macromolecule composed of at least two kinds of amino-acid containing structures associated through intermolecular - S - S - bonds. It is possible to conceive of the gel-like macromolecules as being built up from these two kinds of structures that differ only in their size, their content of total amino acid and in the frequency with which the acidic amino acid and cystine molecules occur in their amino acid-containing moiety and possibly their carbohydrate content and composition. One of the two amino-acid containing structures can be identified, after solubilization of the sparingly soluble material, as the typical water-soluble blood-group specific glycoprotein already well characterized and containing relatively small amounts of aspartic and glutamic acids and cystine. The second kind of structure m a y be similar to the first in overall structural pattern, but contains alarge proportion of total amino acids and especially of the acidic amino acids and cystine, or it may be composed of amino acids alone. An intermolecular - S - S - bonded association of these two types of molecule could give rise to a very large macromolecular structure that, because of its size, would most probably have low solubility and would presumably break up into its components when treated with simple reducing agents such as thioglycolate and sulphite. So far the second kind of structure has not been isolated as a homogeneous molecular species. All preparations obtained however have shown considerable group specific activity, which indicates that this second structure m a y contain some specific determinant carbohydrate groups and thus be glycoprotein in character, Only a few sparingly soluble glycoproteins have been examined, and these show certain differences in behaviour with reducing agents and in chemical composition. Until the appropriate data obtained from a larger number of preparations is available, the suggestions brought forward in this paper must be considered tentative. There is evidence that the overall size and conformation of the group specific glycoproteins influence the extent and firmness with which they combine with the corresponding antibody. I t is possible therefore that much useful information will be forthcoming to explain these and other observations if it is known more exactly how the very large and sparingly soluble glycoproteins differ from those that are of smaller molecular size and are readily soluble in water.
ACKNOWLEDGEMENTS
One of us (J.R.D.) thanks the Royal Society and the Nuffield Foundation for the award (1962-1963) of a Commonwealth Bursary. We thank Dr. A. PUSZTAI, Dr. J. M. CREETH and Dr. WINIFRED WATKINS for much useful discussion and advice. REFERENCES I 2 3 4 5
D. AMINOFF,W. T. J. MORGAN AND W. M. WATKINS,Biochem. J., 46 (195o) 426. R. A. GIBBONS, W. T. J. MORGAN AND M. N. GIBBONS, Biochem. J., 60 (1955) 428. A. PUSZTAI AND "~V. T. J. MORGAN, Biochem. J., 80 (1961) lO 7. W. T. J. MORGAN AND H. K. KING, Biochem. J., 37 (1943) 640. A. PUSZTAI AND W. T. J. MORGAN, Biochem. J., 80 (1961) lO 7.
Biochim. Biophys. Acla, IOI (1965) 3oo-314
314 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20
J. R. DUNSTONE, W. T. J. MORGAN
M. N. GIBBONS, Analyst, 80 (1955) 268. C. J. M. RONDLE AND W. T. J. MORGAN, Biochem. J., 61 (1955) 586. N. NELSON, J. Biol. Chem., 153 (1944) 375. G. E. FRANCIS,\¥. MULLIGAN AND A. ~VORMALL,Isotopic Tracers, The Athlone Press, London, ISt Ed., 1959, p. 277. E. J. CoNwAY, Microdiffusion Analysis and Volumetric Error, Crosby-Lockwood, London, 4th Ed., 1957. L. WARREN, J. Biol. Chem., 234 (1959) 1971. M. DUBOlS, K. A. GILLES, J. K. HAMILTON, ['. A. REBERS AND F. SMITH, Anal. Chem., 28 (1956) 35 ° • M. P. TOMBS, F. SOUTER AND N. F. MACLAGEN,Biochem. J., 73 (1959) 167. \¥. T. J. MORGAN AND H. IK. KING. J., 37 (1943) 640. t2. A. GIBBONS AND W. T. J. MORGAN, Biochem. J., 57 (1954) 283. R. IR. RACE AND R. SANGER, Blood Groups in Man, Blackwell, Oxford, 4th Ed., 1962. S. MOORE, D. H. SPACKMAN AND "W. H. STEIN, Anal. Chem., 3 ° (1958) 1185. A. PUSZTAI AND x~V.T. J. MORGAN, Biochem..]., 88 (1963) 546. E. SCHRAM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. A. PUSZTAI AND \¥. T. J. MORGAN, Biochem. J., 93 (1964) 363.
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