Equilibrium and kinetic studies of oxygen binding to fragments of Lymnaea stagnalis (freshwater snail) haemocyanin obtained by proteolytic digestion.

Functional fragments of the haemocyanin from the gastropod mollusc Lymnaea stagnalis (freshwater snail) were obtained by partial digestion with trypsin and plasmin. The fragments were purified by ion-exchange chromatography and characterized by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Three types of single-functional unit fragment were isolated from the trypsin digest, and two immunologically distinct three-functional unit fragments and a single-functional unit fragment were isolated from the plasmin digest. The O2-binding behaviour of the fragments was investigated by equilibrium and kinetic methods. Over the pH range 7.0-8.2, in the presence of 10-20 mM-CaCl2, all of the single-functional unit fragments displayed non-co-operative O2 binding and showed no evidence of a Bohr or a salt effect. A Hill coefficient of less than 1.0 was obtained with one of the two three-functional unit fragments studied, whereas both of these fragments displayed a Bohr effect. Functional heterogeneity of the fragments was indicated by the variation in the O2 affinity, the P50 (partial pressure of O2 at half saturation) ranging between 0.26 and 0.77 kPa (approx. 2-6 mmHg). Stopped-flow data reflected the O2 equilibrium behaviour. Thus there was a fall in the value of the O2 dissociation rate constant from approx. 15 to 1s-1 in parallel with the increase in O2 affinity.