Exp. Path., Bd. 14, S. 40-54 (1977) Clinical Chemistry Department of the TCtenyi Street Hospital, Budapest, Hungary

Theinfluence of inorganic phosphate and citrate anions on the effect of glycosaminoglycans during cQllagen fibril-formation By M. NEMETH-CSoKA With 15 figures (Received January 2, 1976) Key words: phosphate- citrate; anions; glycosaminoglycans; collagen fibril-formation; fibrillogenesis; chondroitin sulfate; hyaluronic acid; keratan sulfate; heparin; heparitin sulfate

Summary Some anions like phosphate, sulfate and citrate delay the fibril-formation. The interaction of phosphate and citrate with chondroitin sulfate-A (CSA) in binding to collagen was investigated in different environmental conditions. By changing the concentration of phosphate from 5 to 50 mM/l and that of CSA from 0.5 to 16 mM/l some kind of competition of the anions was discovered. When different equilibration, systems were used the affinity of CSA to collagen was found to be 10 times greater than that of phosphate at pH 7.2 and 1= 0.15. As phosphate anions bind to collagen at physiological concentration. phosphate anions may be supposed to influence the biological fibril-formation. On the other hand the CSA exchanges phosphate for collagen. Therefore, glycosaminoglycans (GAG) in connective tissue ground substance in fibrillogenesis may regulate the ion binding and through this the tendency of aggregation of the collagen molecules. The binding of citrate to collagen and its effect and possible role in fibril-formation was also evaluated. Abbreviations CSA = chondroitin sulfate-A CSB = chondroitin sulfate-B CSC = chondroitin sulfate-C GAG = glycosaminoglycans HA = hyaluronic acid HEP = heparin HS = heparitin sulfate KS = keratan sulfate-1 P = inorganic phosphate

The effect of glycosaminoglycans (GAG) in collagen fibril-formation is known to be influenced by many factors like the preparation of collagen (WOOD 1960a, 1960b, WOOD and KEECH 1960), the molecular weight (NEMETH-CSOKA 1961) and the degree of sulfatation of GAG (NEMETH-CSOKA 1974a), the ionic environment etc. (GROSS and KIRK 1958) and these could be the reasons for some misinterpretations in the literature. In earlier papers we explained the effect of GAG on fibrillary aggregation as due to thermodynamic processes: fibril-formation is an entropy-driven process and GAG are to facilitate the gain of entropy due to fibrillary aggregation (NEMETH-CSOKA 1974 b). The cations and anions as has been recognized by several authors (BENSUSAN and HOYT 1958, CANDLISH and TRISTRAM 1963, GROSS and KIRK 1958, HARRINGTON 1958, RUSSELL and COOPER 1969) are influencing the rate of fibril-formation from collagen solution. Recently we have found that among the anions phosphate and citrate retarded the collagen fibril-formation and this delay can be counteracted by chondroitin sulfate-A (CSA). The aim of the present paper is to conduct a systematic study on the interaction of phosphate and citrate with CSA binding by collagen in special regard to fibril-formation.

40

111aterial and methods For studying the fibrillary aggregation the heat gelation of collagen was used according to BENSUSAN and HOYT (1958). Collagen was extracted by acetic acid from rat skin and after salting out collagen protein was dissolved in Tris orland phosphate buffer of 0.02-0.05 Mil and of pH 7.2. The gelation of neutral salt soluble collagen was carried out in thermostated Helma cuvettes. The turbidity during precipitation was measured at 560 nm. The effect of all known GAG (two series of international reference standards, see figures): hyaluronic acid (HA), chondroitin sulfate-A, B, C (CSA, CSB, CSC), keratan sulfate-1 (KS), heparitin sulfate (HS), heparin (HEP), was investigated in different ionic environments. CSA for investigation of ions interaction was extracted from bovine nasal septum at O°C by 2 % of KOH, according to the method of EINBINDER and SCHUBERT (1950). Its sulfate-S content was 5.5 %, the molecular weight 14,000. The CSA content was measured on the basis of its hexuronic acid content by the modified carbazole method of BITTER and MUIR (1962). The estimation of inorganic phosphate (P) was done by reduced molybdenate technique and that of citrate by the method of SAFRAN and DENSTEDT (1948). The interaction of anions was studied by measuring the distribution of anions at equilibrium by equilibration in a dialysis system according to ~ACGREGOR and BOWNESS (1971) and by a buffer-agarose gel equilibration system according to OBRINK and WASTESON (1971). The in vitro fibril-formation was carried out by the gelation technique as reported earlier (NEMETH-CSOKA 1974a). The concentration of GAG was expressed in mMolfl disaccharide content based on the hexuronic acid content.

Results The reference GAG standards differently influenced the kinetics of the fibrillary aggregation relative to control untreated collagen (figs. 1 and 2). CSA increased whereas HS and HEP delayed fibril-formation, the size order of the effect depends on the ratio of collagen to CSA as it was postulated earlier (NEMETH-CSOKA 1974a). x

THE EFFECT OF GLYCOSAMINOGLYCANS

ON

THE FIBRIL -FORMA TlON

0.0. at 560 nm CSA

1,5 . HA

1,0

HS

0,5 pH~

I

7,25

= 0,16

Collagen' GAG. 10 ' 1

o ~~:"-::::::::::::::::~------+-------t----+--';;' 1,0 30 20 10 o x

Reference from

min

standards

K. MEYER

Fig. 1. The effect of glycosaminoglycans on the fibril-formation of collagen. Gelation of collagen solution (2 mgt ml protein content) at 27°C in the presence of GAG of 0.2 mg, ml concentration at pH 7.2 and 1= 0.16. The optical density of the solution is plotted against the time in minutes. (GAG are reference standards from K. MEYER, Columbia Univ. New York.)

41

x

THE

EFFECT OF GLYCOSAMINOGLYCANS

ON

FIBRIL-FORMATION

0.0. at 560 nm CSA

1,5

csc

HS

1,0

pH- 7,25 I - 0,16 Collagen, GAG - 10 ' 1

0.5

~~:- _ _- - - - - - - - - H E P

= __-+-

o o

- - - - ~

30

60

min

x

Referenc;e standards

from

14. B. MATHEWS

Fig. 2. The effect of glycosaminoglycans on the collagen fibril-formation followed by gelation technique, at ionic strength 0.16. (GAG are reference standards from M. B. MATHEWS and coworkers, University of Chicago.) GAG influence fibril-formation differently. THE

EFFECT OF GLYCOSAMINOGLYCANS AT

HIGH

0.0. at 560 nm

ON FIBRIL FORMATION

IONIC STRENGTH

esc

1,5

1,0

Buffer +NaCI

pH. 7,24 0,5

I

1&

0,50

phosphate buffer of 0,0514+ NaCI

0 1 l l L . - - - - - - l - - - - - - - + - - - - - - 4 - - - - - - - - l - - - 0 > min

o

10

20

30

Fig. 3. The effect of glycosaminoglycans on collagen fibril-formation followed by gelation technique at high ionic strength (I = 0.5). GAG increased uniformly the rate of fibril-formation.

THE

EFFECT

OF ANIONS

ON

FIBRIL-FORMATION

O.D. at 560 nm

1,5

1,0

pH.

7,2~

I .0,2

0,5

Tris buffer of 0,05 M

~Citrat/-)

O~~=======min o 10

20

30

Fig. 4. The effect of different anions (in sodium salt from) on the rate of fibrillary precipitation of collagen. Collagen is dissolved in Tris buffer of 0.05 molarity. The content of added anions is 0.05 MIl.

Table 1. The effect of different anions (in sodium salt form) on the growth period of fibril-formation

SCN-

CO.-CI-"

F-

CH 3 COO-

7 7 10 10 10

min min min min min

Tris buffer N03 - -

12 min 12 min 12 min

S2 0 3-S04-P04---

18 min

S03--

Citrate-

22 min 26 min 30 min

1 ml collagen in M 0.5 Tris buffer of pH 7.':2

enhancing effect

no effect

delaying effect

+ 1 ml sodium salt solution of 0.05 mM/1

At high ionic strength (I = 0.5) no difference could be detected among GAG: all increased uniformly the fibril formation (fig. 3). The effect of different anions in sodium salt form were also tested (fig. 4). SCN -, Cl-, F-, CO 2-- enhanced the fibril formation whereas Tris buffer, NOs --, SOs -- had no effect but S20S' S04--, P0 4-- and citrate delayed it (see table 1). The order of anions corresponds to the reverse order of the lyotropic series of HOFFMEISTER. The striking retarding effect of phosphate, sulfate and citrate anions could be reversed by adding CSA (see c. g. that of citrate in fig. 5). These results suggested that the possible effect of GAG on fibril-format!on might be achieved indirectly through the competition of anions and CSA, the latter was studied in details.

43

THE EFFECT

OF GLYCOSAMINOGLYCANS DELAYED

BY

ON

FIBRIL -FORMATION

CITRATE

0.D. at .560 nm CSA

1,5

HA

0,006 M

1,0

0,5

_--------HS min

20

10 1= 0,21

30

~o

phosphate buffer of 0,02 M

Fig. 5. The effect of different GAG on the collagen fibril-formation delayed by citrate, added in

0.003 MIl. At higher citrate content the fibrillary precipitation of collagen is inhibited.

The binding of P and CSA to collagen was studied in 2 different ways: a) P binding was measured at different P concentrations and in rising CSA content. b) CSA binding by increasing the CSA content and as the function of rising P content. In both cases with and without NaCl of 0.2 molarity. as to a) 100 mg of Cl-free collagen were suspended in 3 ml phosphate of pH 7.2,1-50 mM/l, for 3 days at O°C with and without NaCl of 0.1 molarity. 98 % collagen remained undissolved. The binding of P to undissolved collagen was calculated from the decrease of P content of the supernatant and expressed as bound P in f.lM/g collagen. In order to investigate the effect of CSA, it was added to the above reaction mixture in 16 mM/l concentration. The bound CSA was given in the same manner in f.lM/g collagen. Each experiment was carried out fourfold and the figures represent the means of four replications. The binding of P to collagen gradually increased from 55 f.lM/g to 155 f.lM/collagen with the rising of the molarity of phosphate buffer. In the presence of chloride the bound P became greater by 20-30,uM/g. The bound CSA to collagen was 45f.lM/g at a P content of 1 mM/l and decreased gradually by rising of the P content down to 9f.lM/g. Cl bound to collagen at 0.1 molar solution in 440 f.lM/g quantity and it also decreased by rising of the P content (see fig. 6). as to b) The binding of CSA to collagen was investigated by the same system as that of P. In each reaction mixture of 3 ml, 100 mg collagen were equilibrated with different anions and the anions' uptake of the undissolved collagen was measured. Collagen bound 5-140f.lM/g CSA by increasing the CSA concentration from 3 up to 32 mM/1. The binding of P at 5 ruM/l concentration was 7-9f.lM/g, at 10 mM/115-52,uM/g. Thus there was an inerease in the P binding to undissolved collagen as the function of the rising CSA content. The binding of Cl did not change significantly by increasing the CSA content (see fig. 7).

44

THE

BINDING OF

P,

CI AND CSA

OF COLLAGEN A T pH

7. 2

P pMol/g Collagen 200

I

CSA

100

40

pMol!g Collagen

20

P mMOI!1

pMOI/9 Collagen

P

CI

CSA

4°1

100

pMoIJ9 Collagen

20 CSA

1 5

10

.--- P _____ CI

Collagen' buffer z 10, 1

25

50

P mMol/1 phosphat buffer 100 mMol/1 NaCl

~P Q---{]

CI

JI--I(

CSA

Collagen' CSA

z

10 ' 1

Fig. 6. The binding of inorganic phosphate (P), chloride (Cl) and chondroitin sulfate-A (CSA) of collagen (undissolved) expressed in mM/g collagen protein at different P contents (1-50 mM/l) with and without CSA of 16 mM/1 with(below) and without (above) 100 mM/1 of NaCI. The ordinates represent the bound P, Cl and CSA, the abscissas show the P content of the reaction-mixtures.

45

THE

Jlfvfoljg

Collagen

P

CSA

BINDING

OF P AND

CSA

OF COLLAGEN

Jlfvfol/g P

AT pH

7.2

Collagen

CSA

50 1,00

200

"'--+--__4---_---_

CSA

10

30

20

_CSA

--P Phosphate

10

mfvfOI/1

0---0 CI

buffer

Phosphate

10 mfvfol/I + 160 mfvfolll

CSA

30 mfvfOI!1

20

buffer

5 mtvtOI/I

+ 160

NaCI

mfvfol/I NaCl

Fig. 7. The binding of inorganic phosphate (P), chloride (Cl) and chondroitin sulfate-A (CSA) of collagen (undissolved) expressed in ,uM,g collagen protein at 5 mM/l (right) and at 10 mM/1 (left) P content of the reaction-mixtures. The ordinates represent the bound P, Cl and CSA, the abscissas show the CSA content of the reaction-mixtures in mM/1. THE

EQUILIBRATION

mfvfol/I

OF ~HOSPHATE AND AT pH

CSA

7.2

20

10

"-- 1 10

20

6,2 mMol/1

13 mfvfol!1

A 1,5 mtvtoll

A 2,5 mMol I

•

o

CSA

P

.Fig.8. Partition of phosphate (P) and chondroitin sulfate-A (CSA) at equilibrum in a system where the inner compartment (upper part) contains CSA in different concentrations and dialyzed against the solvent phosphate buffer of 8 mM!1 at pH 7.2 (lower part). The ordinate represents the P and CSA content in mM/1. The empty column shows the P content, the striated column the GSA content. The numbers under the columns indicate the concentration of CSA in mM!I, and the difference of P content between the two compartments (Ll values).

46

THE EQUILIBRATION OF PHOSPHATE

AND

CSA IN BUFFER-AGAROSEGEL

SYSTEM IN THE PRESENCE OF COLLAGEN P

(pH 7.2)

CSA

mMol/1

5

o

0 --

t

---- t t ---- t

buffer agarosegel

5

Collagen

CSA

P

mg%

5

o

0 --

t

buffer agarosegel

5 mMol/1

o

Phosphate

.CSA

•

Collagen

Fig. 9. Partition of inorganic phosphate (P) and chondroitin sulfate-A (CSA) in a buffer-agarose gel system (upper part) and its change on the effect of collagen protein (2 mg/ml) dissolved in the buffer phase (lower part). The ordinates represent the content of P and CSA in mM/1. The black columns indicate the collagen content, the striated ones the CSA and the empty columns that of P content. The arrows indicate the direction of the change.

Equilibration dialysis between P and CSA CSA dissolved in phosphate buffer in a semipermeable compartment was dialysed against the solvent buffer at 0 °C for 48 hours. CSA could not pass through the- membrane. At equilibration a difference occurred in the P content: namely in the compartment of CSA P became less with 1.5 and 2.5 mM/I at CSA contents of 6.2 and 13 mM/1. At the same time the total cation content in the compartment of CSA increased by 3.8 and 6.3 mM/1 respectively. - The decrease of the P content was less than calculated on the basis of CSA surplus in the mM/1 content (see fig. 8). Partition of P and CSA between collagen solution and agarose gel Agarose gel was prepared by cooling 3 ml of hot agarose solution of 2 % w/v in 25 ml Erlenmeyer flasks. Collagen (2 mg/ml) dissolved in phosphate buffer was layered in the same volume on the top of the gel discs and the system was equilibrated at 0 °C for 4-5 hours. Parallel series with dissolved CSA were run at the same time. CSA and P could freely distribute between collagen and gel. As collagen remained (or most of it, 80-95 %) the above

47

system seemed to be suitable for investigation of affinity and partition of P and CSA for collagen. The CSA content increased in the buffer phase containing dissolved collagen in comparison with buffer initially of the same CSA content but without collagen in it, whereas that of P changed reversely. As the P content of the buffer decreased by 5 mM/l as a result of 0.5 mM/l CSA, it may be supposed that the affinity of CSA to collagen is 10 times greater than that of P (see fig. 9). In other words the binding of CSA to collagen increased from 10 to 135 f-lM/g collagen as the function of the rising CSA content of the solution from 0.3 mMjl to 1.6 mMjl and at the same time the release of P from collagen changed from 15 to 105 f-lMjg collagen respectively (see fig. 10). CHANGE CF BINDING CF CSA AND P TO COLLAGEN THE EFFECT OF INCREASING CSA CONTENT ( buffer- agarose gel

equilibration

J

CSA binding pMIg Collagen

100

(+J

a F-=======~;:::'---:':~--------o:-':>--'-------~ 1,0

1,5

mWI CSA

(-)

100 pH= ~2

I pMIg Collagen P binding

T

= 0, 11

= a_4°C

P = 10mMII

CI .100mMII

Fig. 10. The change of bouna chondroitin ~111ht,e-A (CSA) and phosphate (CSA) and phosphl1te (1') in p,M/g protein of dissolved collagen in the buffer/agarose gel system (see fig. 4) and the effect of the increase of the CSA content of the buffer. Upper part: the increase of bound CSA to collagen, lower part: decrease of bound P to collagen, both given in p,Mjg collagen.

Fig. 11. Change of the rate of fibril-formation in different buffers. The effect of inorganic phosphate (P) and chondroitin sulfate-A (CSA) content on the rate of fibril-formation. The numbers at the end of the curves represent the collagen (CSA) quotients at a collagen content of 2-3 mg/m!. The ordinates show the optical absorbance of collagen sol-gel, the abscissas show the time given in minutes. Fig. 12.. The change of phosphate (P) content between collagen gel and its superabundance on the effect of chondroitin sulfate-A (CSA) without and with NaCl in the system. The empty columns with continuous line represent the P contents: the lower ones are the P content of gel, that of the superabundance are behind.

48

THE EFFECT

OF ELECTROLYT - CONTENT ON THE RATE OF COLLAGEN

0,05 M phosphate buffer

0,05M NaCI 0,01""

Collagen ' CSA 10: 1

1,0

PRECIPITA TlON

phosphate buffer

1,0 10, 0,1 10'0,3 10'0,0

10:0,3 10:0,1

0,20

0,20

10:0,0

__+-min

~~=+::=-';""_-+ 5 10 15

a

-=:::""---+-_ _+-_--1-_ _' - min

a

20

0,05 M Tris buffer

5

15

10

0,05 M Tris

20

bu ffer

0,01 M phosphate buffer

I,D

~::::::E:;::§~

1,0

10:1 10 : O;()

Collagen' CSA

0,20

0,20

Fig. 11

..>-----