Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus).
The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2'-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.
Department of Chemistry, University of Richmond, VA 23173.
R
R
Topham
B
B
Cooper
S
S
Tesh
G
G
Godette
C
C
Bonaventura
J
J
Bonaventura
D000818:Animals; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006458:Hemolymph / Q000378:metabolism*; D006737:Horseshoe Crabs / Q000378:metabolism*; D007501:Iron / Q000097:blood*; D033862:Iron-Binding Proteins; D007526:Isoelectric Point; D008970:Molecular Weight; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D002614:Chelating Agents; D033862:Iron-Binding Proteins; D033863:Transferrin-Binding Proteins; D007501:Iron