Occurrence of Collagen in the Phylum Mollusca

No. 4621 May 24, 1958 NATU RE ing evidence is produced. We wish to emph&Bize that all the species mentioned above were described long after Simpson's publication and that we only feel that it is necessary to record this correction because of the frequent uncritical references of other authors to the Brora. Coal. It may still be proved that there were pre-Cretace ous angiosperm s, but this particular evidence must be discarded. N. F. HUGHES Sedgwick Museum, Cambridge . R. A. COUPER New Zealand Geological Survey. March 20. 'Simpson, J, B., Nmure, 139, 673 (1987). ' Balme, B, E., C.S.I.R.O. Fuel Research (Australia), Ref. TC25 (1957). • Erdtman, G., <hd. F6ren. Sl.ockh. Plkh., 70, 265 (1948) . 'Couper, R. A., New Phytol., 56, 280 (1956). Table 1. Species 1483 OoCURRENCE OF HYDROXYPROLINB IN THREE SPBCIES OJ' M:oLLUBOA Percentag~ in dry, Ash ash-free material (percentage Total Hydroxyln dry nitrogen material) prollne Sample Whole animal Body-wall Viscera Mytilus Whole animal t4ulis BYBBU8 apparatus Ctenldla Mantel Loli/lo oulgaris wall Fin Cranial cartilage Headfoot• Helix a.spersa Hydroxyproline nitrogen/ to~! nitrogen (percent) 1·30 2·16 7 ·75 9 ·5 13 0·94 1·91 Trace 8 0·48 10·3 0·49 1·50 0·60 8 ·4 8·0 1 ·9 0·80 23 11 0 ·43 1·06 16·1 11 ·5 0 ·29 1·00 12 4·15 9 ·3 4·80 0·82 12·5 0·70 16 • One arm together With associated part of head Occurrence of Collagen in the Phylum Mollusca CONSIDERABLE interest has recently been shown in the occurrence of a collagen type of protein among 1 2 the various invertebrat e groups. X-ray diffraction , studies have explored a wide range of examples, and more recently chemical investigatio ns have used various chromatog raphic techniques to determine the compositio n of certain preparation sa- 5 • Most of this work has been performed on tissues from single species bearing little or no relationshi p to one another. In the present study, the occurrence of collagen has been investigate d in representat ives of the three major classes of the phylum Mollusca, using the occurrence of hydroxypro line as an indication of the presence of ·a collagen-ty pe protein. The examples were: class Gastropoda , Helix aapersa (garden snail); class Lamellibra nchia.ta, Mytilus e,dulis (edible mussel); class Cephalopod a, Loligo vtdgaris (common squid). The dead animals were we.shed in isotonic saline for several days, rinsed with distilled water and blo~ted dry with filter paper Moisture content, which was close to 85 per cent for all the samples, was estimated by drying to constant weight at 105° C. and the ash was measured by igniting at 550°. Hydroxypr oline was determined by a modification• of the Neuman and Logan' procedure and total nitrogen by Kjeldahl determinat ion. Results have been expressed in terms of the ratio ofhydroxy proline nitrogen to total nitrogen. Analyses were performed on the whole snail and mussel and on the parts of the animals indicated in Table 1. Determinations on the squid were carried out on various regions and not on the whole animal. The byseus apparatus and ctenidia were dried in ·vacuo over phosphorou s pentoxide before analysis was carried out. The results in Table I show the presence of hydroxypro line and hence of collagen in almost all samples examined, with a tendency to be concentrated into specific structures. This is particularl y shown for the snail, where the collagen is concentrate d in the body-wall. About 240 gm. of mussel bodies were extracted with hot water at pH 4·5 using higher temperatur es for ea.ch successive extract (60-90° C.). The material containing hydroxypro line went preferentia lly into solution, the mean hydroxypro line nitrogen to total nitrogen ratio (as per cent) being 0-76 for the extracts. Approxima tely 50 gm. of snail bodies (dry weight about 8 gm.) were soaked for a short time in 5 per cent hydrochlor ic acid, followed by nwnerous changes of ½ per cent sodiwn chloride both before and after removal of the viscera, for a week. Preliminar y experiment s had shown that a short treatment of the snail body-wall with saturated lime water increased the rate of extraction of hydro:x:yproline-rich material in warm water. Four changes of lime water were given during four days. The body-wall material was washed and extracted at 70° C., 2 hr., so• C., 2 hr. and 90° C., 2 hr. The first extraction was lost in an unsuccessfu l attempt to fractionate it. The second and third extracts were filtered and concentrat ed and dried over phosphoru s pentoxide. The second extract was observed to set to a gel at 4° C. after concentrati on. The analytical results for these extracts are given in Table 2. Table 2. ANALYSIS Extract Yield (mgm.) 2 112 200 3 01' EXTRAO'.l'S FR0H LnfED SNAIL BoDY·WALLS Ash (per cent) Hydroxyproline 4·0 3·6 Total nitrogen Hydroxyprollne nitrogen/ total nitroiten (percent) 16·6 17 ·8 6·95 7 ·6 Percentage in dry, ash-free material 10·8 12·6 It was noted that the hydrolysa.t e of both extracts was light in colour and resembled that for gelatin samples known to be largely free of polysaccha ride. The hydro:x:yproline and total nitrogen figures are therefore likely to be close to those of the intact collagen of the snail body-wall. I am indebted to Dr. A. Courts and Mr. A. A. Leach for valuable discussion. s. C. MELNICK The British Gelatine and Glue Research Association , 2a. Dalmeny Avenue, Holloway, London, N.7. March 27. 'Bear, R. S. , "Adv. Protein Chem.", 7, 69 (1952). • Rudall, K. M., Symposia Soc. Exp. Biol., 9. "Fibrous Protein• and Their Biological S!gnUlcance", 49 (Camb. Univ. Press, 1966). • Wataon1 M. R., "Recent Advances In Gelatin and Glue Beee&rch" ' 179 1Pergamon PreBB, London, 1958). • Saper, J"., and White, W. E., Nahwe, 181, 285 (1958). 'Lenhoff", H. M., Kline, B. S., and Hurley, R., Biocmm. Bioph11• . .Ada, 21, 204 (1967). • Leach, A. A., .AMl. 0/um. (In the presa). 'Neuman, R. G., and Lopn, lit. A., J. Biol. Chem., 184, 299 (1950), © 1958 Nature Publishing Group  (...truncated)