Prevention of tropocollagen aggregation by l -ascorbic acid

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BBA 3 3 1 4 7

Prevention of tropocollagen aggregation by L-ascorbic acid Tropocollagen, the soluble precursor of collagen can be extracted from connective tissues such as skin with neutral salt or dilute citric or acetic acid solutions 1. It is synthesised b y fibroblasts 2 and, under suitable conditions, aggregates extracellularly to form fibrils, possibly by a process of self-assembly3. Vitamin C has been found to prevent or delay this fibrillogenesis. For these experiments a known (approx. o.O5~o, w/v) solution of acid-extracted calf-skin collagen 4, dialysed against 0.05 M acetic acid (pH 3.5) and centrifuged at IOO ooo × g for 60 rain, was equilibrated at 37 °, and mixed with an equal volume of citric acid-Na2HPO 4 buffer (pH 7.4), adjusted to I = 0.23 with NaC1, and the formation of a gel of aggregated tropocollagen was monitored 5 b y recording the percentage transmittance of light at 4o0 nm in a Unicam SP5oo spectrophotometer equipped with a pinhole between the cell and the phototube to minimise the effect of scattered light. A typical sigmoid plot of percentage transmittance against time of the type analysed b y WOODs was obtained. However, no gel was formed in the presence of I mM L-ascorbic acid. With one batch of tropocollagen 4 raM, I mM and 500/~M n-ascorbic acid prevented tropocollagen aggregation for at least 7 days, 2 days and 2 h, respectively. However with a second batch of tropocollagen, 500 #M L-ascorbic acid delayed aggregation for 2 h, whilst in the presence of 200 #M L-ascorbic acid a gel formed after 35 rain at 37 ° compared with IO rain in the control tube lacking ascorbate. Aggregation was not prevented by 200 #M or i mM dehydroascorbic acid, nor by 200 #M GSH. When a mixture of I mM n-ascorbic acid with acid soluble collagen and buffer was dialysed against distilled water a precipitate of aggregated collagen was obtained which appeared identical in the electron microscope with that obtained in the absence of ascorbic acid, except that the diameter of the fibrils m a y have been smaller. When FeSO 4 (final concn., 2 raM) was added to the acid soluble collagen, buffer and ascorbic acid mixture, a similar precipitate of aggregated collagen which also appeared identical in the electron microscope, gradually formed. This precipitation m a y have occurred as a result of the autooxidation of the I mM L-ascorbic acid. The lysis of glycosidic bonds in the presence of low concentrations of L-ascorbic acid 7 is inhibited by the copper-chelating agent 8-hydroxy-7-iodoquinoline-5-sulphonic acid (J. C. CAYGILL, unpublished observations). However it seems unlikely that the antifibrillogenic action of L-ascorbic acid is a result of such lysis because i mM L-ascorbic acid is as effective in the absence of as in the presence of 500 #M 8-hydroxy-7-iodoquinoline-5-sulphonic acid or 200/~M CUSO4"5 H~O, the inhibition is reversible and appears to be instantaneous, at least with some preparations of tropocollagen. With one extract, 200/~M L-ascorbic acid added just as precipitation commenced (i.e. after the "nucleation" phase of W o o d s) was sufficient to prevent fibrillogenisis, whereas with another extract, which had been stored at -- I5 ° for some months prior to dialysis and ultracentrifugation, it was neccessary to add ascorbic acid up to 30 min before adding the citrate-phosphate-NaC1 buffer in order to prevent fibrillogenisis. It was found that different batches of tropocollagen behaved Biochim. Biophys. Acta, 181 (1969) 334-336

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differently with respect to the length of the "nucleation" phase, the rate of aggregation and in susceptibility to ascorbic acid. Similar differences between batches of tropocollagen have been noted by others s,8. Recently a portion of the tropocollagen possibly corresponding to the "nucleus-forming" fraction produced in the lag phase was reported to react with bovine nasal chondroitin sulphate protein. It would be interesting to know whether this portion represents tropocollagen monomers which lack bound ascorbate, while the less reactive non-nucleating fraction represents tropocollagen with which ascorbate is associated by electrostatic or other bonds. It is concluded from these experiments that L-ascorbic acid in vitro delays or prevents the normal aggregation of tropocollagen to form native fibrils under conditions of temperature, pH and ionic strength similar to those in vivo. Furthermore the fibrils produced in vitro under these conditions become insoluble in dilute citric acid solutions 9 in a manner analogous to their maturation in vivo. In tissue culture, fibroblasts actively synthesising collagen require 5°/~g/ml (284/aM) of ascorbic acid in order to prevent a reduction in the rate of collagen synthesis and to prevent morphological changes characteristic of scorbutic animals 1°. Dehydroascorbic acid does not replace ascorbic acid in tissue culture n and does not prevent collagen aggregation in this system. Fibroblasts from scorbutic animals show an altered morphology, including the appearance of numerous intracellular vacuoles and changes in the endoplasmic reticulum ~. Collagen synthesis is decreased but not prevented TM. Intracytoplasmic collagen fibrils have been reported in a number of human mesenchymal tumour cells which were rapidly synthesising collagen 1~. It is possible that in the absence of ascorbic acid collagen synthesis proceeds normally, but at some stage, possibly that of hydroxylation of proline or lysine or both, it acquires the ability to self-assemble and will do so unless ascorbic acid is present to prevent this. Hence in the absence of ascorbate overall collagen synthesis may be depressed as the newly synthesised or hydroxylated collagen begins to precipitate and accumulate intracellularly in fibroblasts. One can imagine that aggregation of collagen, possibly still attached to ribosomes or protocollagen hydroxylase14, may lead to morphological disturbances. It is therefore suggested that L-ascorbic acid (vitamin C), which prevents or delays collagen fibrillogenesis under conditions likely to obtain in connective tissue in vivo, may be required by fibroblasts to inhibit intracellular aggregation of collagen, and that regulation of removal of L-ascorbic acid by diffusion or oxidation may be of importance in regulating extracellular collagen deposition. I would like to thank Mr. A. C. Cooper for generously providing tropocollagen solutions, and Dr. J. A. Chapman, Mr. J. Bard and Mr. S. Grundy for the electron microscopy.

Rheumatism Research Centre, University of Manchester, Manchester (Great Britain)

J . c . CAYGILL*

I K. A. PIEZ, in G. N. RAMACHANDRAN,Treatise on Collagen, Vol. I, Academic Press, New York, 1967, p. 2o 7. Present address: Tropical Products Institute, 56/62 Gray's Inn Rd., London, W.C.I, England. Biochim. Biophys. Acta, 181 (1969) 334-336

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2 R. R o s s , in B. S. GOULD, Treatise on Collagen, Vol. 2A, A c a d e m i c Press, New York, 1968, p. i. 3 J. A. CHAPMAN, in G. E. W. WOLSTENHOLME AND M.O'CoNNOR, Principles of Biomolecular Organisation, Churchill, L o n d o n , 1966, p. 129, 4 D. S. JACKSON AND IL. G. CLEARY, in D. GLICK, Methods of Biochemical Analysis, Vol. 15, Interscience, New York, 1967, p. 25. 5 G. C. WOOD, in D. A. HALL, International Reviews of Connective Tissue Research, Vol. 2, A c a d e m i c Press, New York, 1964, p. I. 6 G. C. WOOD, Biochem. J., 75 (196o) 598. 7 J. C. CAYGILL, Biochim. Biophys. Acta, 17o (I968) I. 8 B. P. TOOLE AND D. A. LOWTHER,Biochem. J., lO 9 (1968) 857. 9 J. GROSS, Nature, 181 (1958) 556. io J. J. REYNOLDS, Exptl. Cell Res., 47 (1967) 42. 11 Y. SHIMIZU, D. S. McCANN AND M. K. KEECH, J. Lab. Clin. Med., 66 (1965) 659. 12 B. S. GOULD, in B. S. GOULD, Treatise on Collagen, Vol. 2A, A c a d e m i c Press, L o n d o n , 1968, p. 323 • 13 R. A. WELSH AND A. T. MEYER, Arch. Pathol., 84 (1967) 354. 14 D. J. PROCKOP AND t~. I. KIVIRIKKO, in ]3. S. GOULD, Treatise on Collagen, Vol. 2A, A c a d e m i c Press, L o n d o n , 1968, p. 215.

Received December 3rd, 1968 Biochim. Biophys. Acta, 181 (1969) 334-336

BBA 33148 Dissociation studies on the toxin of Clostridiurn botulinum type B It has been shown that a major toxic product present both intracellulafly z and in lysates ~ ofClostridium botulinum type B is a macromolecular structure with s == 15. We have reported the presence of lysates of low molecular weight toxic moieties s. It has recently been shown that this I5-S toxic component can be dissociated under alkaline conditions and the dissociation products isolated b y chromatography on DEAE-cellulose 4, one product possessing approx. 5 times the specific activity of the original product, and designated by the authors as the Ba component. This report constitutes a study on the chemical nature of these dissociation products in an attempt to determine whether two distinct protein species are present in the I5-S material and whether or not a chemical relationship exists between these materials and low molecular weight toxic moieties reported earlier. The methods for isolation of the I5-S toxin have been described previously 1. The pelleted material was dissolved in o.o 5 M acetate buffer (pH 4.5) at 5 mg/ml and yielded a single sharp peak of s = 15.5 in the analytical ultracentrifuge. End group analysis, by the method of GRAY AND HARTLEY5 was carried out on this material and only arginine was detected. This is in agreement with end group analysis carried out previously on low molecular weight toxic materials 3. Since only one amino acid was detected no such tests were run on the dissociation products. The I5-S toxin was chromatographed under alkaline conditions b y methods previously described 4, to yield the two major dissociation products, the Ba or highly toxic material, and the other poorly toxic material (designated here Peak I I DEAE). Amino acid analyses were carried out on a Beckman model 12o analyser 6 on the I5-S material, Ba and Peak I I DEAE. Three separate batches of material were prepared, processed and Biochim. Biophys. Aeta, 181 (1969) 336-338