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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
Published on: March 10, 2010
Studies of serum protein complexes with nickel using crossed immunoelectrophoresis
J L Nielsen1, O M Poulsen, A Abildtrup
1Department of Chemistry and Biochemistry, National Institute of Occupational Health, Copenhagen, Denmark.
Insights
Nickel exposure can alter human serum proteins, affecting diagnostic accuracy. This study reveals nickel binds to multiple serum proteins, including albumin and alpha-1-antitrypsin, with potential implications for analytical methods.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Nickel is a metal with potential biological effects.
- Understanding nickel-protein interactions is crucial for diagnostics and toxicology.
- Physiological nickel levels are low, but higher concentrations can occur in certain exposures.
Purpose of the Study:
- To investigate the binding of nickel to human serum proteins.
- To identify specific serum proteins that bind nickel.
- To evaluate immunoelectrophoretic methods for analyzing nickel-protein complexes.
Main Methods:
- Crossed immunoelectrophoresis was used to analyze nickel-protein complexes in human serum spiked with nickel.
- Radioactive 63Ni was employed for autoradiography to identify labeled proteins.
- Rocket immunoelectrophoresis was used to assess nickel-induced precipitation of serum proteins.
Main Results:
- Nickel addition caused dose-dependent changes in electrophoretic patterns of prealbumin, alpha-1-lipoprotein, alpha-1-antitrypsin, and alpha-2-macroglobulin.
- Autoradiography confirmed binding of 63Ni to albumin and alpha-1-antitrypsin, with broader labeling at higher concentrations.
- Nickel induced precipitation of serum proteins, notably IgG, which could interfere with chromatographic analyses.
Conclusions:
- Human serum contains multiple proteins with nickel-binding affinity beyond albumin and alpha-2-macroglobulin.
- Immunoelectrophoretic techniques offer a viable alternative to chromatographic methods for studying nickel-protein interactions.
- Nickel-induced protein precipitation necessitates careful consideration in analytical procedures involving nickel-protein complexes.
Abstract:
Crossed immunoelectrophoresis of human serum spiked with nickel in the range 0.85-24 mmol Ni/L was used to study nickel-protein complexes. These high concentrations, which are far higher than the physiological level (approximately 7.8 nmol/L), were used to saturate both high and low affinity binding sites. Addition of increasing amounts of nickel resulted in dose-dependent changes of the electrophoretic patterns of prealbumin, alpha-1-lipoprotein, alpha-1-antitrypsin and alpha-2-macroglobulin. Radioactive 63Ni was used for crossed immunoelectrophoresis autoradiography experiments for further identification of nickel-protein complexes. When a 63Ni pulse of 740 kBq/application was used, many human serum proteins were labeled. When using a 63Ni pulse of 185 kBq/application only albumin and alpha-1-antitrypsin were visualized clearly. The binding of large amounts of nickel to albumin, visualized by autoradiography, may reflect the high abundance of albumin in human serum as compared to other serum proteins. Addition of nickel to serum proteins resulted in liquid phase precipitation of serum proteins, and rocket immunoelectrophoresis was used to demonstrate that IgG in particular is precipitated. This precipitation of serum proteins may disturb the elution profile when chromatographic techniques are used to analyze nickel-protein complexes. Consequently, immunoelectrophoretic methods may also be attractive alternatives to column chromatographic techniques. The present study demonstrated that, besides the nickel-binding of albumin and alpha-2-macroglobulin, several other serum proteins have nickel-binding affinity.
