The effect of arginine on inhibiting amyloid fibril derived from ß-casein and the binding studies with multi-spectroscopic techniques.

In general, ß-casein is a stable molecular chaperone. However, the fact that amyloid fibrils derived from ß-casein has been reported in some cases, which were usually associated with some malignant breast diseases. As an important amino acid, arginine not only exhibits the significance in casein synthesis in mammary gland, but also has great potentiality in inhibiting the amyloid fibril formation. Therefore, the influence of arginine on the amyloid fibrils formed by ß-casein and further molecular mechanism were studied firstly with multi-spectroscopic techniques in the present work. The results of Thioflavin T determination, particle size analysis, transmission electron microscope observation showed that arginine not only inhibited the aggregation of ß-casein at the growth stage, but also depolymerized the mature amyloid fibrils at the saturation stage. The further fluorescence experiment results demonstrated that the complex was formed between ß-casein and arginine. Besides, there was one binding site and 0.48 nm binding distance. The thermodynamic parameters like ΔG0, ΔS0, ΔH0 were all negative, showing their binding reaction was spontaneous, and hydrogen bond and van der Waals force were the possibly chief intermolecular forces. Furthermore, the synchronous fluorescence spectra showing that the conformation of ß-casein was affected and its tyrosine residues were gradually buried inside the protein. Our research would provide new insights into the treatments for the breast amyloidosis.

Amyloid fibril formation by casein and fatty acid composition in breast milk of mastitis patients.

Mastitis can cause changes in the nutrient composition of breast milk, which may be harmful to both newborns and lactating mothers. In this study we preliminarily evaluated amyloid fibrils formation by casein and fatty acids (FA), as well as their potential relation with each other in the breast milk of mastitis patients. Six healthy volunteers and six mastitis patients were recruited from the Maternal and Child Health Care Hospital in Changchun were enrolled. Amyloid fibril content was assessed by thioflavin T fluorescence analysis, transmission electron microscope, circular dichroism, and proton nuclear magnetic resonance. FA contents were measured by gas chromatography. Healthy breast milk contained no amyloid fibrils but inflammatory breast milk did. Several FAs (hendecanoic acid, myristolenic acid, pentadecenoic acid, eicosatrienoic acid) differed significantly between the two groups (p < .05). The concentrations of the eicosatrienoic acid and eleven carbonic acids in the inflammatory groups were lower than those in the healthy groups, but the myristolenic acid and pentadecenoic acid were the opposite trend. Early detection of amyloid fibrils should be performed in lactating mothers with mastitis. Changes in FAs may reflect the importance of abnormal metabolism in amyloid fibril formation. PRACTICAL APPLICATIONS: The work preliminarily clarified the relationship between inflammation, fibril content, and fatty acid (FA) composition in breast milk. Healthy milk contained no amyloid fibril formed by casein but the inflammatory milk did. FAs were also significantly different between the two groups. Thus, an early determination of amyloid fibrils in milk should be considered for lactating women with mastitis to avoid the further malignant development. Additionally, the changes in FAs may reflect the importance of abnormal metabolism and oxidative pathways in amyloid fibril formation in the breast. Therefore, this study provided foundations for further investigation on the association between inflammation, fibril content and FA composition in breast milk.

The Influence of Ca2+ and Zn2+ on the Amyloid Fibril Formation by ß-Casein.

BACKGROUND: The amyloid fibril formation in different tissues or organs is related to amyloidosis. The Ca2+, Zn2+ and heparan sulfate (HS) are important elements and compositions in human body, which play a key role in regulating various physiological activities. Recently, there are increasing evidence suggest that they are closely linked to the amyloid fibril formation. OBJECTIVE: The effect of Ca2+ and Zn2+ on the amyloid fibril formation by ß-casein was investigated in the absence and presence of HS, which was significantly to explore the relationship between the concentration changes of Ca2+ and Zn2+ and amyloid fibril formation. METHODS: In this work, the influence of Ca2+ and Zn2+ on the ß-casein fibril formation in the absence and presence of HS was investigated by various methods of Thioflavin T fluorescence assay, transmission electron microscopy and intrinsic fluorescence measure. RESULTS: The results demonstrated that Ca2+ and Zn2+ promoted the ß-casein fibril formation. The effect of Ca2+ was greater than that of Zn2+. Meanwhile, the both metal ions had stronger effects when ß-casein was incubated with HS together. In addition, it was also observed that the microenvironment of ß-casein was changed because the intrinsic fluorescence peaks were red-shifted on the influence of Ca2+ and Zn2+. CONCLUSION: Ca2+ and Zn2+ were capable of promoting the ß-casein fibril formation in the both absence and presence of HS. This work set up the foundation for further researching of the amyloidosis pathogenesis and provided new insight for us to understand relationship between the inflammation and amyloidosis.

Interaction of L-arginine with κ-casein and its effect on amyloid fibril formation by the protein: multi-spectroscopic approaches.

Herein, the interaction of l-arginine (ARG) with κ-casein, and its effect on amyloid fibril formation of the protein, have been investigated in vitro by resonance light scattering (RLS), fluorescence, UV-Vis absorption spectroscopy and transmission electron microscopy (TEM) under simulated physiological conditions. The results indicated that ARG inhibited fibril formation by reduced and carboxymethylated κ-casein (RCMκ-CN), and there was interaction between ARG and RCMκ-CN, proved by the observation of enhancement in RLS intensity attributed to the formation of RCMκ-CN-ARG complex. It was also demonstrated that ARG strongly quenched the intrinsic fluorescence of RCMκ-CN through a static quenching mechanism. The corresponding thermodynamic parameters (ΔH, ΔS and ΔG) were tested to show that the binding process was spontaneous and mainly enthalpy driven with an unfavorable entropy, and both hydrogen bond and van der Waals forces played a key role in the binding of ARG and RCMκ-CN. The determined value of the distance r between ARG and RCMκ-CN Trp97 residue evaluated by fluorescence resonance energy transfer (FRET) was 2.94nm. Furthermore, the conformational investigation from synchronous fluorescence showed that the RCMκ-CN Trp97 residue was placed in a less polar environment and more difficultly exposed to the solvent after addition of ARG.