Abstract
For echidna and canine milk lysozymes, which were presumed to be the calcium‐binding lysozymes by their amino acid sequences, we have quantitated their calcium‐binding strength and examined their guanidine unfolding profiles.
The calcium‐binding constants of echidna and canine lysozymes were determined to be 8.6 × 10^6^ M^−1^ and 8.9 X 10^6^ M^−1^ in 0.1 M KCl at pH 7.1 and 20 °C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of α‐lactalbumin, through a stable molten globule intermediate. However, neither calcium‐bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg‐white lysozyme, avian calcium‐binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel‐Boston‐Berlin: Birkhauzer Verlag. pp 9‐31) that the lineage of avian and echidna calcium‐binding lysozymes and that of eutherian calcium‐binding lysozymes diverged separately from that of conventional lysozymes.