The Ca2+ binding properties of calpain are of great interest, both biochemically in the wider context of EF-hand proteins, and physiologically, in the context of calpain regulation. There are two major parameters which one might wish to measure: the actual number of binding sites (n) and the binding constants for Ca2+. The latter is normally the macroscopic binding constant for Ca2+ of the molecule as a whole (Kd), or the microscopic binding constants for each of the EF-hands, but for cooperative binding these are much more difficult to measure. There is evidence of various kinds to suggest that Ca2+ binding causes conformational change in the whole molecule, and this forms the basis for measuring Ca2+ binding by means of changes in fluorescence.