Fig. 1

bLF Effects on Hemozoin Formation, Heme Binding, and Hemozoin Degradation. (a) Inhibition of Hemozoin Formation by bLF. Differential Absorption of Heme (405 nm) and Hemozoin (620 nm) was calculated using the equation depicted in the figure and is denoted as the hemozoin formation unit. Measurements were conducted throughout 0–4 h. MQ acts as a negative control for hemozoin formation. CHQ represents 1 mM chloroquine, a positive control known for inhibiting hemozoin formation. bLF was tested at 0.5, 5, and 50 µM concentrations. All experiments were performed in triplicate. (b) Hem binding assay. The interaction between heme and bLF was examined through co-precipitation. Tubes display the samples pre and post-mixing, followed by centrifugation. The co-precipitate (complex) of heme and bLF is highlighted within the white dotted circle. (c) SDS-PAGE analysis of heme-bound lactoferrin. Sup represents the sample before mixing bLF and heme, while Ppt represents the precipitate formed after mixing bLF and heme and washing twice with PBS. The samples were treated with SDS buffer to release the bound proteins and subjected to SDS-PAGE analysis. The gel shows bands corresponding to bLF in the Ppt lanes, indicating that a significant amount of bLF is bound to heme. Duplicate lanes for both Sup and Ppt demonstrate the reproducibility of the results. (d) Hemozoin degradation by bLF. The degradation efficacy of hemozoin was evaluated by artificially synthesizing hemozoin in tubes, followed by bLF addition. After a 4-h degradation reaction, the tubes were inspected and documented. The contents were separated into supernatant (Sup.) and precipitate (Ppt.), with each examined microscopically. Magnifications were 200× for the supernatant and 400× for the precipitate. Undegraded hemozoin remain as a precipitate, similar to MQ. However, successful degradation results in a heme-derived brown color within the tube. The microscope field of view presents a representative image.