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

Abstract

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 Any required sample was withdrawn and the freezing cycle repeated appropriately.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 Pso 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 AlogPso/ApH with respect to freezing. None of the four haemocyanins exhibited significant dependence of cooperativity (n^) on