Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG) and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG.
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