Presence of functional domains in Limulus polyphemus hemocyanin.
In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit alpha is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments result in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistant domain.
Lello
Z
Z
Department of Biochemical Science, University La Sapienza, Rome, Italy.
Luca
G
G
Maurizio
B
B
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper; D006433:Hemocyanins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D006737:Horseshoe Crabs / Q000378:metabolism*; D008970:Molecular Weight; D008121:Nephropidae / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D003300:Copper; D002248:Carbon Monoxide; D006433:Hemocyanins; D014357:Trypsin; D010100:Oxygen