Binding of DNA to albumin and transferrin modified by treatment with water-soluble carbodiimides.
N-Acylurea derivatives of albumin and transferrin prepared with the water-soluble carbodiimides N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide and N-ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide have been found to bind different types of DNA. The two proteins were reacted with varying amounts of carbodiimide in water at pH 5.5 for 36-60 hr at 20 degrees, and then purified. In the case of iron-loaded transferrin, reactions with carbodiimides were in phosphate-buffered saline (pH 7.5) to prevent loss of iron from the protein. [3H]N-Ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the modified proteins, and gel electrophoresis for changes in charge and possible aggregation through cross-linking. Binding of DNA to N-acylurea proteins was studied by means of gel electrophoresis and nitrocellulose filter binding. N-Acylurea albumin and N-acylurea transferrin at low concentrations retarded the migration of lambda-Pstl restriction fragments, pBR322 plasmid and M13 mp8 single-stranded DNA on agarose gels, while at higher concentrations of modified protein the N-acylurea protein-DNA complexes were unable to enter the gel. Nitrocellulose filter assays showed that binding pBR322 DNA and calf thymus DNA to N-acylurea proteins is rapid and dependent on protein concentration and the ionic strength of the medium. N-Acylurea albumins prepared with each each of the two carbodiimides gave comparable plots for DNA bound versus protein concentration. On the other hand, binding of DNA by N-acylurea transferrins differed according to the carbodiimide used in the synthesis. N-Acylurea CDI-tkransferrin (prepared with tertiary carbodiimide) was less effective than either of the two N-acylurea albumins in binding DNA. In contrast with these results, N-acylurea Me+-CDI-transferrin (prepared with quaternary carbodiimide) was far more effective in binding DNA and in this respect was similar to the N-acylurea albumins. On the basis of experiments in which N-acylurea protein-DNA complexes were treated with heparin, two types of binding could be distinguished. These were a weak binding occurring in the initial stages of interaction and a tight binding which developed on further incubation of the complexes. These studies show that binding of DNA by N-acylurea proteins is a reversible process dependent on ionic strength; interaction appears to be electrostatic in nature, although other forms of binding might be involved.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001667:Binding, Competitive; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D003101:Collodion; D004247:DNA / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology*; D005374:Filtration; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D014168:Transferrin / Q000378:metabolism*
D002234:Carbodiimides; D012709:Serum Albumin; D014168:Transferrin; D012965:Sodium Chloride; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D003101:Collodion; D006493:Heparin; D004247:DNA; D005022:Ethyldimethylaminopropyl Carbodiimide