Interactions of recombinant human lactoferrin with deoxyribonucleic acids

The present study investigated the ability of recombinant human lactoferrin (rhLF), isolated in pure form from the milk of transgenic goats, to nonspecific interactions and specific complex formation with natural DNA and synthetic DNA fragments. Intact rhLF was utilized, containing a europium ion in the metal-binding center possessing long-lived fluorescence. In a microplate system, rhLF-Eu³⁺ interacted with adsorption-immobilized total double-stranded DNA from the calf thymus with Ka = 1.7  10⁶ M^–1. In the same heterophase system, singleand double-stranded (ds-) oligonucleotides with specific (L) or nonspecific (S) sequences were bound in solution to rhLF–Eu³⁺ and competitively inhibited the interaction of the labeled protein with DNA on the solid phase. The inhibitory effect was contingent upon the specificity, structure, and length of the nucleotide sequence, exhibiting a decrease in the following sequence: ds-L₂₅ > ds-S₅₀ > ds-S₂₁₅ > ds-S₁₈₀ > ds-S₁₁₅ > L₂₅ > S₅₀. In an alternative system, specific (ds-L₂₅) and nonspecific (ds-S₅₀) oligonucleotides, biotinylated through a long mobile linker, were immobilized onto the solid phase coated with biotin-binding protein streptavidin. The affinity of rhLF for these ligands was characterized by Ka of 6.4  10⁷ and 7.1  10⁶ M^–1, respectively. The binding to the nonspecific nucleotide sequence was also much more sensitive to the ionic strength of the solution and the concentration of an inert protein. The ability of rhLF to interact with DNA would confirm the identity of natural and recombinant proteins and encourage the use of rhLF as a biologically active food supplement and pharmaceutical substance.

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