Purification and characterization of fish liver ferritins

Ferritin from the liver of fresh, salt and brackish water fishes was purifi ed by thermal denaturation of liver homogenate followed by ammonium sulphat e fractionation and Sephacryl S-300 gel filtration. Yield and iron content of purified fish ferritins were 0.016-0.026 mg/g of wet tissue and 4-14%, r espectively. The iron content of ferritins from marine and brackish species was higher than from fresh water species. The phosphate/iron ratio ranged from 0.5 to 1.8 and was higher than mammalian ferritins. The fish ferritins have 5-6% neutral carbohydrate. Native gel electrophoresis and molecular w eight analysis revealed the presence of a monomeric ferritin. SDS-gel elect rophoresis and immunoblotting showed a single protein band of 21 kDa sugges ting the presence of similar sized subunits in the native structure of fish ferritins. Isoelectric focusing revealed microheterogeneity with five to s even bands of pi values between 4.1 and 7.0. Variations in the amino acid c omposition were observed. Proline and arginine were not detected in murrel and salmon species, respectively. High proline and low tyrosine contents we re recorded for perch ferritin. Immunological studies by non-competitive in direct ELISA revealed varying degrees of cross-reactivity. Mammalian ferrit ins exhibited a moderate cross-reactivity with anti-fish ferritin. On the c ontrary, very low or no cross-reactivity was observed between fish ferritin and anti-mammalian ferritin. Ferritins from bony fishes such as murrel and rohu exhibited a high degree of cross-reactivity with anti-shark ferritin. However, a moderate cross-reactivity was observed between shark and anti-m urrel ferritin. Ferritin from marine bony fishes, salmon and mackerel and p erch (brackish) showed a low to very low cross-reactivity with both the ant isera. (C) 1999 Elsevier Science Inc. All rights reserved.