Effects of Freezing on the Function and Association State of Crustacean Haemocyanins

The addition of protease inhibitors and antibiotics provides a valuable means of stabilizing proteins in solution but this is less desirable if the physiological properties of a biological fluid, such as haemolymph, are to be accurately evaluated. Freezing haemolymph has proved to be a convenient way both of transporting samples from remote sites and of storage for longer periods. Although many investigators have suspected and intimated that freezing may affect haemocyanin function there are few data describing exactly what these effects may be. Mangum (1983a) provided some data for Squilla empusa haemocyanin indicating that freezing did not alter the P₅₀ affinity but did reduce the cooperativity of O₂ binding. Similar results were reported for Palaemon elegans (Bridges et al. 1984; Morris et al. 1985). These reports that cooperativity may be reduced by freezing indicate that structural changes occur that may lead to changes in affinity, despite the P₅₀ remaining unchanged.

The haemocyanins from two macruran and two brachyuran species were investigated; Homarus vulgaris (Selbie) and Cancer pagurus (L.) from Europe, together with Hemigrapsus nudus (Dana) and Cambarus diogenes (Girard) from North America.

The effect of freezing on the affinity and cooperativity of oxygen binding was determined by the construction of oxygen equilibrium curves for fresh, native haemolymph and for the same haemolymph subjected to a known number of freezing cycles. The freezing protocol consisted of flash freezing (–80°C) the haemolymph in small samples which were kept frozen for 30 min before thawing at room temperature. Any required sample was withdrawn and the freezing cycle repeated appropriately. Oxygen equilibrium curves were constructed at 15°C as described by Bridges et al. (1984) or Morris et al. (1988). Both methods are spectrophotometric and directly comparable. Oxygen affinity was analysed as the P₅₀ in dependence of pH and the cooperativity was calculated for the same equilibrium curves (Fig. 1). Using a fiducial limit of P = 0·05, covariance analysis showed no significant change in ΔlogP₅₀/ΔpH with respect to freezing. None of the four haemocyanins exhibited significant dependence of cooperativity (n₅₀) on pH. Subjecting the n₅₀ data to Student’s t-test showed, however, that freezing one or more times significantly reduced cooperativity. Importantly, the most significant effect was after freezing once only. Subsequent freeze cycles elicited a relatively small decrease in the value of n₅₀. A reduction in cooperativity with no change in P50 describes an increase in the value of P₇₅ and a corresponding decrease in the P25 value. Affinity changes at oxygen tensions away from the P₅₀ are then more significant. The magnitude of the effect of freezing was directly related to the initial value of nso, being large in H. vulgaris but small in C. diogenes.

Changes in the association state of non-frozen haemocyanin and material frozen a known number of times were investigated using fast protein liquid chromatography (FPLC, Pharmacia) with the appropriate columns (Fig. 2). Calibration markers were ferritin, aldolase, ovalbumin and chymotrypsinogen A. The fresh haemolymph was withdrawn from the animal, passed through a Millipore filter to remove particulate material and applied to the column within 30min. Frozen haemolymph was prepared as described previously.

Haemocyanin is normally present in a number of association states in the haemolymph but the relative proportions vary both between species and with changes in the haemolymph microenvironment (reviewed by Mangum, 1983b). It was clear in all cases that a single freeze and thaw had a significant effect on haemocyanin association, ranging from merely noticeable in C. pagurus to dramatic in H. vulgaris. In all four species the pattern was for the fully associated dodecameric haemocyanin (approx. M_r830000) to decline; and for smaller hexameric (M_r415000) haemocyanin and monomers (M_r70000) to increase (Fig. 2). Further freezing exacerbated the effect in all cases.

Reduced temperatures (>0°C) leading to a progressive dissociation of the haemocyanin was suggested as a possible cause of changes in the affinity of haemocyanin from Callianassa californiensis (Miller & Van Holde, 1974), although in re-evaluating these data Miller & Van Holde (1981) suggest that uncontrolled temperature changes may make these data suspect. Similar significant cold-induced decreases in cooperativity have also been associated with marked oxygen affinity changes in the haemocyanin of the two holarctic crabs Hyas coarctatus and H. araneus, where the changes may have some physiological significance (S. Morris & C. R. Bridges, in preparation). These reductions in cooperativity differ from those reported in the present study in the important respect of being reversible. The effect of freezing on crustacean haemocyanins appears to be irreversible.

In conclusion, the present study demonstrates three facts. First, if the native properties of the haemocyanin are at issue the effect of freezing must be carefully determined in each case. Second, the dependence of cooperative oxygen binding on the higher association states of haemocyanin was apparent in all four species. Third, since some intertidal species occasionally experience extracellular freezing or near freezing, the data presented here may indicate that haemocyanin is irreversibly damaged, requiring synthesis of new pigment.