Very Stable High Molecular Mass Multiprotein Complex with DNase and Amylase Activities in Human Milk



Svetlana E. Soboleva1, Pavel S. Dmitrenok2, Timofey D. Verkhovod1, Valentina N. Buneva1,3, Sergey E. Sedykh1, and Georgy A. Nevinsky1,3


  1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Lavrentiev Ave. 8, Novosibirsk 630090, Russia
  2. Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia
  3. Novosibirsk State University, Pirogova Ave. 10, Novosibirsk 630090, Russia


For breastfed infants, human milk is more than a source of nutrients; it furnishes a wide array of proteins, peptides, antibodies, and other components promoting neonatal growth and protecting infants from viral and bacterial infection. It has been proposed that most biological processes are performed by protein complexes. Therefore, identification and characterization of human milk components including protein complexes is important for understanding the function of milk. Using gel filtration, we have purified a stable high molecular mass (~1000 kDa) multiprotein complex (SPC) from 15 preparations of human milk. Light scattering and gel filtration showed that the SPC was stable in the presence of high concentrations of NaCl and MgCl2 but dissociated efficiently under the conditions that destroy immunocomplexes (2 M MgCl2, 0.5 M NaCl, and 10 mM DTT). Such a stable complex is unlikely to be a casual associate of different proteins. The relative content of the individual SPCs varied from 6% to 25% of the total milk protein. According to electrophoretic and mass spectrometry analysis, all 15 SPCs contained lactoferrin (LF) and α-lactalbumin as major proteins, whereas human milk albumin and β-casein were present in moderate or minor amounts; a different content of IgGs and sIgAs was observed. All SPCs efficiently hydrolyzed Plasmid supercoiled DNA and maltoheptaose. Some freshly prepared SPC preparations contained not only intact LF but also small amounts of its fragments, which appeared in all SPCs during their prolonged storage; the fragments, similar to intact LF, possessed DNase and amylase activities. LF is found in human epithelial secretions, barrier body fluids, and in the secondary granules of leukocytes. LF is a protein of the acute phase response and nonspecific defense against different types of microbial and viral infections. Therefore, LF complexes with other proteins may be important for its functions not only in human milk.

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200-400 ml of milk was collected under sterile conditions from women using a breast pump between 4 and 20 days after delivery. The samples underwent purification and stable protein complexes (SPCs) were separated from the other milk proteins. Using a MALDI-TOF mass spectrometry that had been modified with a CovalX HM1 detection system, SPC proteins were analyzed to help determine whether or not fresh human milk LF is able to form stable protein complexes with other milk proteins. . Prior to analysis, intact SPCs from multiple donors were subjected to gel filtration. After gel filtration, 2 μl of the reaction mixture was combined with 2 μl of 0.2% trifluoroacetic acid and 2 μl of matrix (saturated sinapic acid in a mixture of 0.1% acetonitrile and trifluoroacetic acid (1:2)). 1 μl of this final mixture was spotted onto a MALDI plate before being air dried and analyzed. From this analysis, the researchers were able to determine that all 15 SPCs contained lactoferrin and α-lactalbumin while lesser amounts of human milk albumin and β-casein were found.


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