Thermal transitions in the collagenous tissues of poikilothermic animals

J. Thermal Biolm3.~ 1977. Vol. 2, pp. 89 to 93. pergamon Press. Printed in Great Britain

THERMAL TRANSITIONS IN THE C O L L A G E N O U S TISSUES OF P O I K I L O T H E R M I C ANIMALS B. J. RIGBY C.S.I.R.O., Division of Textile Physics, 338 Blaxland Road, Ryde, N.S.W., 2112, Australia

(Received 3 September 1976; accepted 30 October 1976) Abstract--l. The molecular collagen of all animals exhibits a helix-coil transition in physiological solution at a temperature Ta, characteristic for the species. 2. We have found that the collagenous tissues of poikilothermic animals differ from those of homeothermic animals in having another thermal transition at a temperature Ts, where TA< Ta. TA however, is observed only in the native tissues, and not in molecular collagen. Thus, whereas T~ is an intramolecular transition, Ts appears to be intermolecular. 3. Ta for all collagens, correlates with the upper limit of the animals preferred temperature range (body temperature for homeotherms) and TA for the animals so far examined correlates reasonably well with the lower limit. Tm~ RELATIONbetween the thermal properties of a collagenous tissue such as tendon or skin and of the isolated collagen molecule which can be extracted from these tissues is an interesting one. In the latter case the molecule under examination is in contact only with water and whatever ions have been introduced into the water medium, whereas in the native state the molecules within fibrils are under the influence of not only water and ions, but to the extremely specific interaction of a majority of like neighbours as well as to those due to small amounts of unlike neighbours such as polysaccharides, non-collagenous proteins and lipid. In dilute solution all the evidence indicates that the isolated molecule undergoes only one thermal transition when heated--a first order phase change over a small temperature range in which the triplestranded helix collapses to a random-coil structure. This transition, is usually characterised by the temperature, Tv, defined as the midpoint of the change in the property being monitored e.g. optical rotation. It is a function of the chemical composition of the surrounding medium (yon Hippel. 1967). When native tissues are heated, the most obvious and best-known thermal transition which occurs is that referred to as thermal shrinkage (temperature Ts). During this transition, for example, mammalian tendon in water shrinks to about one third of its native length. This phenomenon is a manifestation of the co-operative melting of molecules packed into highly organized super-structures i.e., the melting of crystalline collagen. It is a similar process to the one which occurs in dilute solution, with the exception that the temperature at which it takes place, Ts, is greater than To (by amount 22°C), due to the extra thermal stability gained by molecular interaction (Harrington & yon Hippel, 1961). If sensitive techniques are employed to monitor physical changes in tissue as it is heated (e.g. dilatometry, calorimetry, stress-relaxation), small transitions are observed well below Ts (Flory & Garrett, 1958; Rigby et aL, 1959; Mason & Rigby, 1963; Haly & Snaith, 1971). These axe close to To (as determined in dilute solution), and one is forced to conclude that even in native tissue melting 89

of small groups of molecules, perhaps at the edges of crystallites and in disordered regions of the structure, can begin around Tv. It appears then, that for a wide range of collagens from different phyla the only thermal transition which is manifest in the native tissue (i.e. when maintained in its natural environment) is the first order phase transition of the fundamental molecular unit manufactured by the fibroblast. This transition occurs at Tn in the case of isolated molecules in solution, or near Ta in the native material when sensitive techniques are used. It occurs at Ts in the native material when a similar process occurs, now co-operatively, involving large numbers of molecules. Recently however, we have found another thermal transition, which occurs at temperatures below Tn, and which so far has shown up only in native tissues of poikilotbermic animals (Rigby & Robinson, 1975; Righy & Prosser, 1975); we have never observed it in tissues from homeothermic animals, nor has it been reported in dilute solution studies of collagen extracted from poikilothermic tissues. We originally reported this transition in the cuticle of two different species of earthworm and in the collagenous tissue of jellyfish. For these three animals the new transition, which we designated TA, occurred close to the lower limit of the animal's thermal preferendum, while Tn occurred at the upper limit. The correlation of Tv with the upper limit is already well documented (Rigby, 1971). An interesting difference which will be elaborated upon later is that Ts marks the beginning of an expansion of the sample, whereas Ts and Tv are associated with a contraction. In this paper we present some new results on additional species and discuss some of the structural and biological implications. M A T E R I A L S A N D METHODS

The following collagenous tissues were used: (I) body wall tissue from the earthworms AUolobophora calioinosa and Eiseniafeotida; (2) skins from two closely related frog species Rana temporaria and Rana ridibunda; (3) skin of the carp, Cyprinus carpio; (4) skin of the green sunfish

B. J. RIGBY Lepomis cyanellus. (In this case three samples were in- and to native tissue where it has been detected in volved; one each from groups of fish which had been accli- measurement of Ts. Thus, for 0.154 M NaCl. using mated for 4wks at 5:, 15~ or 25~C. This period of time K = 1.6 (von Hippel, 1967), ( T o - T b ) = -0.25~C. is sufficient for the physiological activity of the fish to Since most physiological solutions are predominantly reach a new equilibrium state (Hazel & Prosser, 1974). The earthworm tissues were prepared in the following 0.154M NaC1, ( T o - T~) for them is still about way. Worms were killed by exposing them to about - 10°C -0.25°C. Even the use of sea water, which is approovernight. After soaking in water at 2OC for a few hours priate for jellyfish tissue, only makes ( T o - T~)= -0.7°C. the cuticles could be removed easily by cutting one end of the animal and shaking the cuticle free of the body. Apart from washing in 0.154 M NaCI, no further treatment Heatin# in HCI at p H l was given to the cuticle. The results for cuticle have been Experiments were also performed in HCI at pHI, described (Rigby & Robinson, 1975}.The worm body when since we have shown elsewhere (Rigby, 1967) that in free of cuticle was cut longitudinally, washed thoroughly this medium collagenous tissues swell, and then melt with 0.154 M NaCl and then treated successivelywith 0.1% abruptly at a temperature at or near To. Furthermore trypsin, 1~,~, NaCI and saturated NaH,PO4. Prepared in this way the body-wall tissue gave a high-angle X-ray dia- the transition at TA can still be observed, showing gram showing strong collagen reflections. It is not claimed that it is independent of pH. that this tissue is entirely free of other non-collagenous protein and polysaccharides. Before the treatment however RESULTS AND DISCUSSION there was a strong ~-protein pattern in the X-ray diagram originating presumably in muscle proteins. Since the force vs temperature curve has the same The frog skins had no treatment other than the removal characteristics for all samples, we have reproduced of any adhering flesh and the outer keratinous layer. Like- only two examples. These are results (shown in Fig. wise, the fish skin had only flesh and scales removed. Thus 1) for the skin of the frog R. temporaria; the upper all the tissues can be described as native, predominantly collagenous materials. The samples were tested in 0.154 M and lower curves are for the sample heated in 0.154 M NaCI, except where otherwise stated; the justification for NaC1 and HC! at pHI, respectively. In the latter test using this medium is given at the end of this section. the sample was mounted in the apparatus in a slack Except in the case of earthworm cuticle thermal transitions condition at I°C. Swelling proceeded and was allowed were determined by the method of temperature induced to continue to equilibrium, as judged by a constant force changes. In this method the sample is held at one force when the sample was slightly extended. Heating end by movable jaws and at the other by jaws attached was then begun, in this case at O.I:C rain -t. The to a force transducer. While in a slack state it is immersed initial force on a sample was generally less than 0.5 g in the test fluid and the temperature reduced to l oC. A and the force change much less, consequently there small extension is then applied to the sample sufficient was vibrational noise superimposed on the recorder to result in a force which will allow changes due to temperature to be detectable. These forces are of (he order trace and the curves shown represent the mean value. of 0.5 g wt in samples of approximate cross-sectional area The three transitions TA, To and Ts are clearly shown I mm2 and produce only small reversible deformations in and the double arrows on the curve after To and Ts the structure. (See also Rigby & Robinson, 1975). After indicate that the force will continue to increase. the force has reached equilibrium (overnight), heating is The mean results for all samples are collected in commenced at a rate of 0.I°C min -t or 0.5°C rain -t. Table 1. The table also contains values of T,, and Heating rates as high as I°C min -t do not affect the T~ reported elsewhere, as well as preferred temperaresults. Transitions are indicated by changes of slope in ture ranges of the various animals. the force-temperature record. Before discussing the results in detail, we briefly summarize the situation with respect to thermal transitions in collagenous tissues. First, all native tissues undergo a first-order melting when heated in aqueous COMPARISON OF RESULTS IN 0.154 M NtCI WITH solution, the familiar thermal shrinkage which occurs OR WITHOUTK÷, Ca2+ AND Mg2÷, AND IN WATER at temperature Ts. This is not a true thermodynamic It is difficult to get precise values for the ionic com- melting temperature; such a determination would position of the tissue fluids of the animals we have require the co-existence of crystalline and amorphous used. However, to a first approximation 0.154M (melted) collagen. Second, if sensitive techniques are NaCI is a reasonable choice (Prosser, 1965). Experi- used, native tissues exhibit a small iransition well mentally, we have not found any significant differ- below Ts in the vicinity of To. Finally there now ences between the transition temperature when appears to be a third thermal transition which as yet measured in water, 0.154 M NaC1 and various mix- has only been observed in tissues from poikilothermic tures of 0.154M NaCI with K +, Ca 2+ and Mg 2÷ animals, and which for reasons to be given later, ion. In fact this is what is to be expected according should only be found in such tissues. to the following empirical equation, which has been shown to fit data for a wide range of collagens and ionic environments (yon Hippei, 1967). RELATIONSHIP BETWEEN TsAND Ta viz. Tn - T°D = ~ KiCi The relation between Ts and To has already been -

,

i

where To is the melting temperature of a given collagen in dilute solution at a salt concentration Ct; T~ -~ To for the same collagen at Ct - 0, and Ki is the molar effectiveness of the salt in question. This relation applies equally well to dilute solution, gels

remarked upon in the Introduction where it was stated that they are temperatures marking the two extremes of the melting of molecular collagen. Ts marks the co-operative melting of large numbers of molecules arranged into crystallites, whereas To

Thermal transitions in the coliagenous tissues

,

0.3

j

0-2 _z

om 0.'1

o

,h

£

3h

,h

TEMPERATURE °C

Fig. 1. Two force-temperature experiments for the skin of Rana lemporarin. The upper curve is for skin immersed in 0.154 M NaCI, and the lower for skin in HCI at pHI. TA, To and Ts are indicated.

marks the melting of single or small groups of molecules. The values of To reported here agree well with determinations of To in dilute solution where these are available. Our value of 23° for earthworm cuticle is compared with 22° (Maser & Rice 1962; Josse & Harrington 1964). These authors used optical rotation and viscometry methods. Andreeva (1970) quotes a value of 32°C for To for R. ridibunda while we find 29°C. Our value of To for carp skin is 29°C, which agrees with that reported by yon Hippel (1967). We know of no solution studies on the body wall of earthworm or Lepomis cyanellus. THE TRANSITIONAT Ta This transition is more difficult to interpret than the one at To. To begin with it appears to be peculiar to collagenous tissues from poikilothermic animals: we have never observed a transition below To in tissues from a wide range of homeothermic animals, in-

cluding those of the mammalian parasitic worm Ascaris. Apart from the tissues from animals used in the present study, we have observed TA and To in tissues of three other poikilotherms of quite different biology, i.e., deep water eel, Alepocephalus and a moray eel, Gymnothorax (Rigby & Prosser, (1975) and a jellyfish Aurelia coerulea (Rigby & Hafey, 1972). For a number of reasons we think that the transition at TA is intermolecular, rather than intramolecular. The first two reasons are based upon experiments with earthworm cuticle in which the length of the freely suspended sample was measured as the temperature was increased (Rigby & Robinson, 1975). The same reasoning applies to the results obtained by the force-temperature method reported in this paper: a drop or increase in force representing an increase or decrease in length of the sample, respectively. First if it were an intramolecular melting (as in the transition at To), we would expect that a contraction of the sample would take place, whereas in fact it

Table 1. Two thermal transitions TA and To which occur in collagenous tissues of poikilothermic animals, together with the preferred temperature range of the animals Collagenous tissue

T,t

To

Preferred temperature range, °C (reference)

8

23

10-23 (Grant, 1955)

12

23

16--23 (Grant, 1955)

13

25

13-26 (Ushakov, 1964)

13

29

18-28 (Ushakov, 1964)

16

29

16-25 (Lankiewicz, 1964)

13 13 13

28 28 28

17

27

17-25 (Rigby & Hafey, 1972).

18

28

18 + (Rigby & Prosser, 1975).

6

18

Taken at 4-6 (Rigby & Prosser, 1975).

A. calioinosa, cuticle & body wall

E. foetida, cuticle & body wall

R. temporaria, skin

R. ridibunda, skin

Cyprinus carpio, skin

Lepomis cyanellus, skin Fish Acclimated at 5° 15° 25°

Aurelia coerulea, bell tissue

Gymnothorax (Moray Eel) skin

Alepocephalus (Deep Water Eel) Skin

To is known to be an intramolecular transition, but TAis thought to be intermolecular in origin. A counterpart of TAhas not been observed in the tissues of homeothermic animals.

92

B.J. RIGBY

lengthens (Rigby & Robinson, 1975). This points to some secondary bond relaxation between molecules and/or microfibrils (Veis & Yuan, 1975; Miller & Parry, 1973). Second, it is completely reversible; not only is the sample length recoverable upon cooling below TA, but the temperature Ta is unchanged upon reheating (Rigby & Robinson, 1975). In view of the specific aggregation properties of soluble collagen, (Hodge, 1967) this reversibility is not surprising. Third, there is no evidence from optical rotation and viscometry studies with dilute solutions, of any transition in poikilotherm collagens other than that denoted by To. The finite width of the transition indicates, of course, that some parts of the molecule are less stable than others, but there is no distinct twostage melting, separated by 10°C or more (von Hippel, 1967; Maser & Rice, 1962; Josse & Harrington, 1964). The fact that the transition occurs under conditions of both neutral and acid pH suggests that ionic interactions are not involved. Some possibilities are hydrophobic interactions or transitions in non-collagenous proteins or polysaccharide. An intriguing point is that TA and To are identical for both the cuticle and body wall collagens of earthworm, yet they are different tissues in so many ways. The former is secreted by epidermal cells and the latter by fibroblasts (Rudall, 1967); they have quite different amino-acid compositions (Fujimoto & Adams, 1964; Rigby 1968); and the body wall collagen exhibits the 600-700 A repeat while the cuticle collagen does not (Rudall, 1967). Further, while there is disagreement about the length of the molecule of cuticle collagen, Maser & Rice (1962) report a mean length of 5400 A and Josse & Harrington (1964) give a value of 9400 £, it is certainly longer than the accepted value for vertebrate collagen of 2800 A. BIOLOGICAL CONSIDERATIONS

Ts is not of biological interest since such temperatures are never reached in the animal's normal environment. There is much evidence to show, however, that To marks the upper limit of an animal's preferred temperature range (Rigby, 1971), and Table 1 contains some new data which supports this view. We have now shown that there is another thermal transition (TA) in the collagenous tissues of poikilotherms, occurring at temperature below To, and which in fact coincides reasonably well with the lower limit of the animal's preferred temperature range (Table 1). We have not observed the transition Ta in the tissues of homeothermic animals, and this is reasonable if the transition at TA serves some biological function in the cold-blooded animal or if the structure has some memory of its thermal history. The last point is apparently ruled out by the results obtained on the skin of the fish Lepomus cyanellus. In this case, T,~ was unaffected by holding the fish at temperatures above and below Ta (13°C), for sufficient time to allow the fish to be acclimatized in all other respects. In other words, as in the case of To, the transition at T,t is almost certainly genetically determined. The inevitable question arises: is the transition at Ta in fact part of the mechanism for signalling that lower temperatures are to be avoided by the animal

if possible? One can make the hypothesis that T~ is physiologically significant. (For example, we have shown (Rigby & Hafey, 1972) that the pulse rate of the bell of the jellyfish shows distinct changes in pattern at TA as well as at To). In pursuance of this suggestion it is perhaps relevant that the collagenous tissues of the poikilotherms are in a mechanically reversible, dynamic state within the range TA-" To compared with their state outside these limits. Beyond To the collagen begins to melt, which of course is highly detrimental to the animal; below Ta the tissue becomes mechanically stiffer, but this is not injurious (since no change of phase is involved). Below T,t the animal can survive, although it may not reproduce or carry out normal life functions. In other words not only do TA and To correlate with the upper and lower limits of the animals temperature preferendum, but the mechanical properties of the tissue appear to be most responsive to temperature within this range (e.g. the reversible increase in expansivity beginning at Ta). Since collagen is very abundant in most animals, these structural transitions could well have an important role in the thermal detection apparatus of the animal.

Acknowledgements--The author thanks Professor C. Ladd Prosser for the skin samples from Lepomus cyanellus, Gymnothorax and Alepocephalus, and Prof. B. Ushakov and V. Alexandrov for those of Rana temporaria and Rana ridibunda

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