Mass spectrometric characterization of Limulus polyphemus hemocyanin

Electrospray ionization-mass spectrometry of ion-exchange and reverse-phase purified hemocyanin from Limulus polyphemus revealed six predominant isoforms with molecular weights ranging from 71,730 to 72,695 Da. The heaviest of these agreed closely with the molecular weight calculated for the previously determined Edman sequence with substitution of isoleucine for valine at position 9 and inclusion of three internal disulfide bonds and one copper atom. Proposed structures for the other isoforms were made on the basis of the molecular weight measurements. Reverse-phase chromatography can be used in addition to the traditional ion-exchange step to produce hemocyanin preparations of greater purity that might be valuable for further detailed investigations of the physicochemical properties of these important proteins. The results reflect yet again the value of mass spectrometry for recognizing molecular microheterogeneity in biological macromolecules and for following protein purification.