Isolierung und Analyse eines Bindungsglobulins für Herzglykoside aus Rinderblut

Abstract

In contrast to the general assumption, ouabain and digoxin, which are plant-derived cardiac glycosides, are steroid hormones of the mammals. They regulate the salt and water metabolism. In general, steroid hormones are transported in blood as complexes with steroid binding proteins. Hence it was the intention of this work to identify and to characterize such a binding protein for cardiac glycosidse in bovine blood.

The cardiac glycoside binding globulin (CGBG) identified in a partially purified serum preparation by affinity labeling with the protein-reactive digoxigenin derivative N-hydroxysuccimidyl-Digoxigenin-3-O-methyl-aminocaproate (HDMA) was used to immunize rabbits. The antibodies raised were immoblilized to prepare an affinity column. This was an essential step to purify CGBG 900,000-fold from bovine blood serum. Purification was performed in 3 days. It included the following steps: Ammonium sulfate precipation (50%), CM-Sephadex cation exchange chromatography, affinity chromatography on an anti-CGBG Sepharose Fast Flow column, anion exchange chromatography on Phenomenex BioSep DEAE. The protein yield of pure CGBG was 0.0001 %

The isolated cardiac glycoside binding protein shows a band at 90kDa on SDS-PAGE. Its N-terminus is blocked by pyroglutamate. The amino acid sequence ALPNVETSV was determined when CGBG was digested with pyroglutamatase. Other sequences obtained were KDLESHYLFERH (digestion with Lys-C) and GKLFNIVNKXQQAL (hydrolysis with CNBr). All sequences were not found in data banks (SwissProt and TREMBL). CGBG contains N-glycosidic bound carbohydrates which contribute to 10-15% of the molecular mass.
The IEP of the native protein is between pH 5.6 and 4.4. Deglycosilated protein shows only one spot at 75kDa and an IEP at pH 5.6 in 2D-Electrophoresis. The calculated mass from size exclusion chromatography is 820 kDa for the native and 180 kDa for the denatured protein. It is concluded that native CGBG circulates as an pentadimer of 90 kDa monomers that are covalently dimerized by disulfide bonds

The intrinsic tryptophane fluorescence quenching experiment using cardiac glycosides as ligands indicates two binding sites with a high (K_D1 = 2.4 nM) and low (K_D2 = 500 nM) affinity binding site. Polyclonal antibodies raised in rabbits against the CGBG recognise specifically the cardiac glycoside binding globulin in a semipurified preparation of CGBG.