Abstract
The two major DNA-binding proteins (designated DNA-binding protein 1 and DNA-binding protein 2) in human serum have been purified and physically characterized. The two proteins co-purify through an ion exchange chromatographic step and DNA-cellulose affinity chromatography. Subsequently, DNA-binding protein 1 can be precipitated by 40% saturated ammonium sulfate; DNA-binding protein 2 precipitates in the 55% to saturation fraction. From these fractions, the two proteins are isolated by different protocols. Both purified proteins are homogeneous by the criteria of sodium dodecyl sulfate slab gel electrophoresis after reduction and denaturation and by sedimentation equilibrium centrifugation. DNA-binding protein 1 has a minimum molecular weight of 126,000; DNA-binding protein 2, 86,000. Amino acid analyses of the two proteins indicate that both are relatively rich in proline and cysteine and contain little methionine. Both proteins contain carbohydrate. Gel electrofocusing confirms the acidic nature of these proteins. DNA-binding protein 1 exhibits a single band upon isoelectric focusing, but DNA-binding protein 2 appears to be polymorphic, exhibiting three bands. NH2-terminal end group analysis of DNA-binding protein 2 yields two major amino acids. DNA-binding protein 1 is an alpha2-globulin as determined by immunoelectrophoresis; DNA-binding protein 2 is only weakly immunogenic. Neither of the proteins appears to be identical to any previously characterized serum protein.
Highlights
Binding protein 1 and DNA-binding protein 2) in human serum have been purified and physically characterized
The two proteins co-purify through an ion exchange chromatographic step and DNA-cellulose affinity chromatography
Neither of the proteins appears to be identical to any previously characterized serum protein. This laboratory has previously reported the isolation of DNA-binding proteins in human serum using affinity chromatography on DNA-cellulose [1]
Summary
1 rnM EDTA, and 0.1 rnM phenylmethylsulfonvl fluoride. Chromatography -A 250-ml sample of pooled adult human serum (Irvine Scientific Co.) was applikd to g QAE-Sephadex column (11.5 x 20 cm) equilibrated in 10 mM potassium phosphate buffer, pH 6.5. The precipitate was collected by centrifugation for 15 min at 13,200 x g It was resuspended at a concentration of 5 mg/ml in 10 mM potassium phosphate buffer, pH 7.8, containing 50 mM NaCl, and dialyzed overnight against 50 volumes of this buffer. RnM potassium phosphate buffer, pH 7.8, containing 50 mM NaCl; after adsorption of the protein sample, the column was washed with this buffer using 5 ml of buffer/g of DNA-cellulose. MM potassium phosphate buffer, pH 6.8, containing 0.5 M NaCl. The fractions containing the DNA-binding proteins were pooled and dialyzed overnight against 50 volumes of 10 mM potassium phosphate buffer, pH 6.5. The column was washed extensively with this buffer before eluting DNA-binding protein 2 in 10 rnM potassium phosphate buffer, pH 6.5, containing 30 mM NaCl. Protein was measured by the method of Lowry et al Proteins used as standards to calibrate the gels were: unreduced human IgG (150,000); conalbumin (77,000); human IgG heavy chain (50,000); ovalbumin (43,000); human IgG light chain [23,500]; and ribonuclease (14,000)
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