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THERMAL TRANSITIONS IN GASTROPOD COLLAGEN AND THEIR CORRELATION WITH ENVIRONMENTAL TEMPERATURE By Be J, RIGBY* and P. MASON*t

[Manuscript received June 27, 1966]

Summary The shrinkage temperature (Ts) in 0'9% saline of the collagen of a number of marine and terrestrial gastropods living in a wide range of temperature conditions has been examined and found to correlate with the environmental temperature of the source, i.e. the higher this environmental temperature the higher is Ts. With other , particularly vertebrates, Ts is found in turn to correlate with the total pyrrolidine content. However, this does not appear to be the case with gastropods examined, except Helix aspersa. Although T s of the native gastro­ pod collagens ranges from 50 to 60°0, it is reduced to 50°0 for all, except one, after treatment of these collagens with polysaccharases. Amino acid analyses of two of the collagens, including the one which is unaffected by polysaccharases, show a pyrrolidine content appropriate to a T s of only 40°0, according to the relation found for vertebrates. It is suggested that polysaccharides playa large role in the thermal stability of these collagens, helping to assure a T s of 50°0 at least, and higher stability for special purposes.

1. INTRODUCTION There is ample evidence that the thermal stability of many collagens, as measured, for example, by the shrinkage temperature (T s) in O· 9% saline, is correlated reasonably well with the environmental temperature of the source (Gustavson 1956). Furthermore, where chemical analyses of the purified collagen are available, a correlation on the molecular level between T s and proline plus hydroxyproline is uncovered (Burge and Hynes 1959; Piez and Gross 1960). Again it is found by physicochemical techniques (optical rotation, viscosity, etc.) that dilute solutions of tropocollagen molecules undergo a helix~coil transition at a temperature, T D, which is about 23 degC below the value of T s appropriate to the collagen from which the solution was made. T D is thus also correlated with proline plus hydroxy­ proline (Burge and Hynes 1959; Piez and Gross 1960; Josse and Harrington 1964). While T sand T D vary directly with the environmental temperature, T s is usually well above, and T D in the vicinity of, the maximum temperature. This is particularly true of the mammals which are, of course, homeothermic. Here T D is only 2~3 degrees above body temperature. It lies close to the maximum environmental temperature for poikilotherms such as , and to the deep body temperature for mammals and birds. Most of this information has been obtained with vertebrates, notably fish and warm-blooded creatures, where environmental temperatures are reasonably well· defined, e.g. cold- and warm· water fish. Such information is valuable and interesting

* Division of Textile Physics, OSIRO Wool Research Laboratories, Ryde, N.S.W. t Present address: Macquarie University, Epping Road, Eastwood, N.S.W.

Aust. J. BioI. Sci., 1967,20, 265-71 266 B. J. RIGBY AND P. MASON because (1) it helps to elucidate the factors involved in the stability of the collagen molecule and fibril; and (2) it throws some light on evolutionary trends among members of a given phylum. The class of the phylum forms a particularly useful group for the study of the general points raised above. We have examined a number of gastropods from the of the New South coast (Dakin 1953), two from the supralittoral zone, and two land . Their environments thus range from that of a warm sea water to exposed supralittoral and to a fully terrestrial . Of the two land snails, one, Helix aspersa, the common garden , avoids direct exposure to the sun and seeks moist cool situations, while the other, Helicella virgata, is notorious for exposing itself to hot arid conditions by congregating on fence and telegraph posts. The two gastropods from the supralittoral zone, Nodilittorina pyramidalis (common noddiwink) and Melarapha unifasciata (blue australwink), are somewhat similar to Helicella virgata in their ability to withstand heat and dryness, especially the former, which lives in the highest supralittoral zone of all true marine on Australian shores. Molluscs with such abilities to resist desiccation exist throughout the world and, in fact, neither Helix aspersa nor Helicella virgata are indigenous to Australia.

II. EXPERIMENTAL As well as the terrestrial and marine gastropods mentioned above the following were examined:

Melanerita melanotragus (black periwinkle) Subninella undulata (turban shell) Austrocochlea obtusa (common periwinkle) Bembicium nanum (common conniwink) Morula marginalba ( borer) Bellastraea sirius (tent shell) Dicathais orbita (cartrut shell)

The animals were killed by freezing, and the bodies were removed from the shells and stored in sea water prior to use. The aim was to examine the shrinkage temperature of the collagen from these animals, but the choice of sample presented a number of problems. The experimental method uses the principle that any dimensional change, due to heating, in a sample held at a fixed length, causes the application of a force to a force-transducer which is attached to one end of the sample (Rigby 1961). This force produces an electric current which is amplified and recorded on a chart, a sudden deflection of the pen representing a structural transition in the sample. The set-up requires samples about 5 mm in length but the cross-sectional area is not important. Pure collagen of this dimension was not available from these animals although tissue from various parts of the showed evidence of collagen when X-rayed, in particular the 2·86 A arc on the meridian. This arc indicated poor orientation and was accompanied by a meridional arc at 5 A and equatorial spots at 9 A which are characteristic of muscle protein. The muscular lateral wall of the foot of the animal was found to be the most suitable part for our purposes and after purification with trypsin (0·1 % overnight), 10% NaCI, and satura­ ted Na2HP04, a reasonably good X-ray diagram of collagen was obtained free from the muscle pattern, but still exhibiting a strong 4 A amorphous halo. THERMAL TRANSITIONS IN GASTROPOD COLLAGEN 267

Electron microscopy showed the axial repeating pattern of 600-700 A. It was expected that, in common with other collagens which have been examined (Piez and Gross 1959), the samples would contain considerable quantities of polysaccharide (:c:: 5 % or more). In order to test for any effect of these polysaccharides on the thermal stability of these collagens, samples were treated with Helicase (a commercial preparation of digestive juice, obtained from Industrie Biologique Francaise, S.A., before testing. Helicase was not expected to damage collagen as it contains a wide range of polysaccharases, but little or no proteolytic activity (Holgen and Tracey 1950). The conditions of treatment were also mild as far as collagen is concerned (0·1 % solution at pH 5·0 and 20°C). It was not certain of course that all polysaccharides present would be eliminated.

TABLE 1

SHRINKAGE TEMPERATURE OF GASTROPOD COLLAGEN IN SEA WATER

Ts for Ts after Native Purification Species Enviroll1llent Material with Helicase (OC) (OC)

N odilittorina pyramidalis Above high water mark 59 58 M elampha unifasciata At high water mark 56 50 M elanerita melanotragus At high water mark 57 50 A ustrocochlea obtusa Between high and low water mark 53 50 Morula marginalba Between high and low water mark 50 50 Bembicium nanum Between high and low water mark 50 50 Subninella undulata Between high and low water mark 50 49 Bellastraea sirius Low water mark, pools 49 49 Dicathais orbita Low water mark, pools 48 48 H elicella virgata Fence and telegraph posts 59 50 Helix aspersa Shady, moist environment 50 50 ------

Amino acid analyses were made of the collagen from three species: Helicella virgata, N odilittorina pyramidalis, and M elarapha unifasciata. These were chosen because of their extreme and, together with Helix aspersa for which analytical data has been obtained by Williams (1960), allows comparison of four gastropods­ two terrestrial and two marine. They were purified as above, including treatment with Helicase, and were then gelatinized with 0 ·IN HCI. The mixture was centrifuged and the clear solution freeze-dried and the gelatin hydrolysed in 6N HCI. T s for all samples was measured in both 0·9% saline and sea water at a heating rate of 1 degC per minute.

IH. RESULTS AND DISOUSSION The T s values quoted refer to the -first indication of shrinkage; they may be 1-2 degC lower than that for the major shrinkage but a consistent criterion was used throughout. Table 1 lists the mean values of T s in sea water of collagen from all species examined before and after purification with Helicase. It was found that there was no difference in Ts between samples tested in 0·9% saline and sea water 268 B. J. RIGBY AND P. MASON and no difference between native material and material which had been purified but had not been treated with Helicase. However, with one exception, treatment of purified collagen with Helicase did not alter the T s value if this was normally 50°C but lowered to 50°C T s values normally more than 50°C. The one exception is collagen from N odilittorina pyramidalis, the T s of which falls only 1 degC, from 59 to 58°C, on treatment with Helicase.

TABLE 2

AMINO ACID ANALYSIS OF COLLAGEN FROM TWO MARINE AND TWO TERRESTRIAL GASTROPODS

Terrestrial Marine

Amino Acid* Helicella Helix N odilittorina I Melarapha virgata aspersat pyramidalist unifasciata

Lysine 10 8·1 22 25 Histidine 4 2·6 6 4 Arginine 56 50·9 52 51 Aspartic acid 78 66·8 65 65 Threonine 26 27·7 25 26 Serine 54 61·4 43 48 Glutamic acid 91 99·1 101 93 Proline 101 104·1 98 98 Hydroxyproline 98 99·5 78 77 Glycine 327 321·0 301 308 Alanine 47 72·3 66 59 Cystine 0 0 0 0 Valine 24 21·5 26 24 Methionine 1 1·2 10 12 Isoleucine 19 12·1 16 18 Leucine 31 23·5 41 40 Tyrosine 6 8·8 10 11 Phenylalanine 13 9·9 14 17 Hydroxylysine 12 8·2 26 23 Cystathionine 2 0 0 1 Amide 130 46·0 105 100

Polysaccharides (as % of dry wt.) ~3 ~2·5 ~5 * Number of residues per 1000 total residues. t Data from Williams (1960). t Corrected values, assuming 100% recovery of nitrogen.

Table 2 shows the amino acid analyses of collagen from the marine gastropods Nodilittorina pyramidalis and Melarapha unifasciata and the land snails Helix aspersa and Helicella virgata. In these analyses collagen accounted for all of the nitrogen in Helix aspersa and Melarapha unifasciata but only for 80% in Nodilittorina pyramidalis, and in the table values for the latter species are given after correction, assuming 100% nitrogen recovery. It can be seen that the collagens from the two marine species are almost identical as are also the collagens from the two land snails. THERMAL TRANSITIONS IN GASTROPOD COLLAGEN 269

They have the general requirements of a collagen: approximately one-third glycine, and significant amounts of hydroxyproline and hydroxylysine. There is general agreement with the findings of Piez and Gross (1959) for a number of other marine , in that compared with vertebrate collagen they have (1) a smaller

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30 1 I 150 200 250 No. OF PROLINE + HYDROXYPROLINE RESIDUES PER 1000 TOTAL RESIDUES

Fig. I.-Correlation between shrinkage temperature (Ts) in sea water or 0·9% saline and the number of proline plus hydroxyproline residues in the following tissues: 1, rat tail tendon; 2, calf skin; 3, human skill; 4, Helix aspersa body wall; 5, tuna skin; 6, shark (dogfish) skin; 7, teraglin (maigre); 8, earthworm cuticle; 9, codfish skin; 10, Nodilittorina pyramidalis; 11, Melarapha unifasciata; 12, Helicella virgata. Ts values were obtained by the method described in this paper. Imino acid contents for tissues 1,2,4,6,8, and 9 are from Harrington and von Hippel (1961) and Tristram and Smith (1963); for tissues 3, 5, and 10-12, by courtesy of Dr. B. S. Harrap; for 7 (maigre), Burge and Hynes (1959). Arrows indicate how Ts has altered after treatment with Helicase. Helix aspersa (4) and tissues 1-3 and 5-9 remain unaltered. proportion of imino acids; and (2) a larger content of hydroxyl groups (serine, threonine, and hydroxylysine). There is no cystine present, although Melarapha unifasciata contains a trace of cystathionine. The main points of difference between the terrestrial and marine gastropods are that the latter have (1) larger amounts oflysine and hydroxylysine (although the 270 B. J. RIGBY AND P. MASON

ratio is about 1 in each case), leucine, and methionine; and (2) lower amounts of hydroxyproline and serine. Table 1 shows a definite correlation between the environment and native collagen shrinkage temperature. As would be expected Helicella virgata, Nodilittorina pyramidalis, and Melarapha unifasciata have the highest Ts values. The value for M elanerita melanotragus is also high, although this animal does not expose itself to the sun as much as the other three. However, its shell is entirely black, in contrast to the others which are light coloured, and for this reason may require extra thermal stability. T s values for these four species are typical of mammalian collagen, while for the most of the others, including Helix aspersa, T s is close to that of a warm-water marine animal, e.g. tuna fieh skin with T s of 49°C. It may be significant that Ts for all species (except Nodilittorina pyramidalis) is reduced to, or remains at, 50°C, after treatment with Helicase. This temperature, as we have already mentioned, appears to be the upper limit for sea creatures, and since those species which have a normal T s of 50°C are not affected by Helicase it might be thought that the collagen of all these animals is stabilized entirely by proline plus hydroxyproline content up to 50°C but that further stabilization is due to polysaccharide. However, as Table 2 shows, proline plus hydroxyproline content is much lower for N odilittorina pyramidalis and M elarapha unifasciata than for Helix aspersa and Helicella virgata. Further, as Figure 1 shows, Helix aspersa fits the relation between T s and the imino acid content quite well, but on this basis the marine collagens should shrink at about 40°C and Helicella virgata at 50°C. The imino acid contents of the collagen of a number of echinoderms, coelenterates, and porifera studied by Piez and Gross (1959) are also much lower than would be expected according to their environment (they are warm-water creatures, Ts = 50°C). There must therefore be some factor involved in increasing the stability of the collagens of N odilittorina pyramidalis and M elarapha unifasciata from 40 to 50°C. This could be a polysaccharide which is not attacked by Helicase at least in the condition when it is bound to collagen. Whatever the reason, thermal stability of the connective tissue of those gastropods examined appears to be assured up to 50°C and for special purposes may be stabilized above this value by polysaccharides. Finally, taking the maximum sea temperature as 28°C, the T s for sea creatures is approximately 22 degC higher than this value, as it is for all the land animals and birds so far examined (e.g. mammals, Ts = 60°C, body temperature 38°C).

IV. ACKNOWLEDGMENTS We wish to thank Miss Janis Cossill for valuable technical assistance; Dr. B. S. Harrap and Mr. A. 1. Inglis, both of the Division of Protein Chemistry, CSIRO, for chemical analyses; Miss Elizabeth Pope, Australian Museum, and Prof. H. G. Andrewartha, University of Adelaide, for specimens. We are also grateful to Dr. 1. W. McDonald and Dr. B. P. Setchell, both of the Division of Animal Physiology, CSIRO, for drawing our attention to the peculiar thermal habits of these gastropods. THERMAL TRANSITIONS IN GASTROPOD COLLAGEN 271

V. REFERENCES

BURGE, R. E., and HYNES, R. D. (1959).-J. Molec. Biol. 1, 155. DAKIN, W. J. (1953).-"Australian Sea Shores." Ch. 16. (Angus and Robertson: Sydney.) GUSTAVSON,K. H. (1956).-"Chemistry and Reactivity of Collagen." p. 224. (Academic Press, Inc.: New York.) HARRINGTON, W. F., and HIPPEL, P. H. VON (1961).-Adv. Protein Chem. 16, 1. HOLGEN, A., and TRACEY, M. V. (1950).-Biochem. J. 47, 407. JOSSE, J., and HARRINGTON, W. F. (1964).-J. Molec. Biol. 9, 269. PIEZ, K. A., and GROSS, J. (1959).-Biochim. Biophys. Acta 34, 24. PIEZ, K. A., and GROSS, J. (1960).-J. Biol. Chem. 235, 995. RIGBY, B. J. (1961).-Biochim. Biophys. Acta 47, 534. TRISTRAM, G. R., and SMITH, R. H. (1963).-Adv. Protein Chern. 18, 227. WILLIAMS, A. P. (1960).-Biochem. J. 74, 304.