A high-throughput method for the quantification of iron saturation in lactoferrin preparations

Abstract

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A(280/466)). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.

Fig. 1

a SDS polyacrylamide gel electrophoresis of DMV International (left) and Sigma-Aldrich (right) bovine lactoferrin preparations. Lanes were loaded with 2.5, 5, and 10 μg of protein. Central lane was loaded with a molecular weight marker (two bands of 70 and 100 kDa denoted on the electropherogram). b SEC elution profile of DMV International lactoferrin obtained using the Superdex 200 10/300 GL column

Fig. 2

Evaluation of the resolution properties of the MonoS 5/50 GL column used to separate bovine lactoferrin in a two-step purification. a MonoS chromatography of DMV International lactoferrin (the collected fraction highlighted in gray bar). b Fraction obtained from the first ion exchange separation was modified using the methods described in experimental section to get iron-saturated and iron-depleted lactoferrin forms. c Elution profiles of apo- and hololactoferrins from the MonoS column from the second separation: apo-Lf (black), holo-Lf (red)

Fig. 3

Correlation between the A ₂₈₀/A ₄₆₆ ratios and lactoferrin iron saturation calculated from ELISA and ICP-MS measurements

Fig. 4

Effect of citrate buffer pH during dialysis at room temperature on lactoferrin iron saturation. Data were obtained through the measurement of absorbance ratio at 280 and 466 nm and converted to lactoferrin iron saturation using the calibration curve described in previous section

Fig. 5

Comparison of iron saturation of the original lactoferrin preparation (Lf DMV International) and hololactoferrins (holo-Lf) prepared by resaturation with ferric ions of Lf DMV International as well as apo-Lf obtained at various pH values

Fig. 6

Analysis of lactoferrin iron saturation under different conditions. a Influence of the Lf/Fe/NTA ratio on the iron saturation process carried out overnight. b Effect of saturation period on the overall efficiency of this process. Lf/Fe/NTA was kept as 1:4:4