Isolation of an iron-binding protein from the hemolymph of the horseshoe crab (Limulus polyhemus)

The presence of an iron-binding protein in the hemolymph of Limulus was detected by gel-filtration of 59Fe-labeled hemolymph. This protein was easily separated from hemocyanin, the oxygen transport protein that constitutes greater than 90% of the protein of Limulus hemolymph. The amount of the Limulus iron-binding protein (LIBP) in hemolymph samples prepared under sterile and non-sterile conditions was identical. LIBP was purified to homogeneity by ion-exchange chromatography. The molecular weight of purified LIBP was estimated by gel-filtration to be 282,000 + 10,000. SDS-electrophoresis demonstrated that LIBP was an oligomeric protein composed of subunits with a molecular weight of 28,000 +/- 2000. The isoelectric point of LIBP was 6.5 as determined by electrofocusing. No 49Fe was removed from purified LIBP by extensive dialysis with EDTA or 2,2'-dipyridyl. Purified, unlabeled LIBP efficiently sequestered 59Fe in the absence of hemolymph indicating that no other hemolymph factors are required for the incorporation of iron into LIBP. The isolation of LIBP would support the proposal that the development of specific iron-binding and transport proteins was not necessarily coupled to the development of hemoglobin as a means to accomplish oxygen transport