The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA-binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high-affinity DNA-binding site of LF with a series of model-specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K(i)  = 5 mM), deoxyribose 5'-phosphate (K(i)  = 3 mM), and different dNMPs (K(i)  = 0.56-1.6 mM). LF interacted additionally with 9-12 nucleotides or nucleotide pairs of single- and double-stranded ribo- and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single- and double-stranded d(pN)(10) provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG(0)) of -8.5 to -10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K(d)  = 10 nM; ΔG(0)  ≈ -11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)(n) of different sequences and lengths was obtained, giving the K(d) values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA.

It is known that that human serum albumin (HSA) and alpha-lactalbumin (LA) possess DNA-binding sites. Electrophoretically homogeneous HSA and LA containing no canonical enzymes were isolated from human sera and milk. Here we have analyzed for the first time the possibility of DNA hydrolysis by these proteins. It was shown that HSA possesses metal-dependent DNase activity, while LA cannot hydrolyze DNA. Several rigid criteria have been applied to show that DNase activity is an intrinsic property of HSA from human sera and milk. HSA preparations were inactive after their dialysis against EDTA or in the presence of EDTA, but were activated after addition of several external metal ions: Mn > Mg > Ca . The best activation of HSA preparations was observed in the presence of two metal ions: Mg +Ca > Mn + Ca ≥ Mn + Mg . In contrast to DNases having only one pH optimum, HSA preparations demonstrated two well-pronounced optima at pH 5.7-5.9 and 6.9-7.1 as well as a weak optimum at pH 8.4-8.6. These results demonstrate the diversity of HSA in the DNA hydrolysis at various pHs and in activation by various metal cofactors. Possible reasons for the diversity of HSA preparations are discussed. © 2018 IUBMB Life, 70(6):501-510, 2018.