Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties.

Contaminating microplastics can interact with food proteins in the food matrix and during digestion. This study investigated adsorption of chicken egg protein ovalbumin to polystyrene (PS, 110 and 260 µm) and polyethylene terephthalate (PET, 140 µm) MPs in acidic and neutral conditions and alterations in ovalbumin structure. Ovalbumin adsorption affinity depended on MPs size (smaller > larger), type (PS > PET) and pH (pH 3 > pH 7). In bulk solution, MPs does not change ovalbumin secondary structure significantly, but induces loosening (at pH 3) and tightening (at pH 7) of tertiary structure. Formed soft corona exclusively consists of full length non-native ovalbumin, while in hard corona also shorter ovalbumin fragments were found. At pH 7 soft corona ovalbumin has rearranged but still preserved level of ordered secondary structure, resulting in preserved thermostability and proteolytic stability, but decreased ability to form fibrils upon heating. Secondary structure changes in soft corona resemble changes in native ovalbumin induced by heat treatment (80 °C). Ovalbumin is abundantly present in corona around microplastics also in the presence of other egg white proteins. These results imply that microplastics contaminating food may bind and change structure and functional properties of the main egg white protein.

The interplay between copper(II), human serum albumin, fatty acids, and carbonylating agent interferes with Cys 34 thiol reactivity and copper binding.

Cys34 thiol group of human serum albumin (HSA) represents major plasma antioxidant. Its reactivity is influenced by multiple factors. The influence of fatty acids (FA; saturated, mono, and poly unsaturated acids from fish oil) binding to HSA, on copper(II) binding affinity and Cys34 thiol group accessibility/reactivity, in the presence of carbonylation agent (methylglyoxal, MG) was examined. HSA-copper(II) content, thiol group reactivity, and HSA carbonylation level were monitored spectrophotometrically. Changes in HSA were followed by fluorescence spectroscopy and native PAG electrophoresis. FA/HSA molar ratio was screened by GC. Together, binding of copper(II) ions and FA to HSA increase the reactivity of Cys34 thiol group (depending on the type of FA), with constant contribution of copper(II) ions of one-third. Carbonylation of FA-HSA-Cu(II) complexes caused a decrease in the Cys34 thiol group content, accompanied by a decrease in the content of HSA-bound copper. The carbonylation level of guanidine groups was not affected by FAs and copper(II) binding. Fluorescent emission spectra of FA-HSA-Cu(II)-MG complexes showed conformational changes in HSA molecule. Although binding of fatty acids and copper ions caused a significant increase in the thiol group reactivity, Cys34 thiol from FA-HSA-Cu(II) complexes reacted with MG in smaller extent than expected, probably as a consequence of conformational changes introduced by carbonylation. Increase in the percentage of reacted-free thiol groups with MG (due to FA and copper binding) may not seem to be very significant, but it is very important in complex biological systems, where catalytic metal is present.

Binding of enterolactone and enterodiol to human serum albumin: increase of cysteine-34 thiol group reactivity.

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.

Fatty acids binding to human serum albumin: Changes of reactivity and glycation level of Cysteine-34 free thiol group with methylglyoxal.

Fatty acids (FAs) binding to human serum albumin (HSA) could lead to the changes of Cys-34 thiol group accessibility and reactivity, i.e. its scavenger capacity and antioxidant property. The influence of saturated, mono and poly unsaturated, and fish oil FAs binding to HSA on the carbonylation level and the reactivity of HSA-SH and HSA modified with methylglyoxal (MG-HSA-SH) was investigated. Changes of thiol group reactivity were followed by determination of pseudo first order rate constant (k') for thiols reaction with 5,5'-dithiobis(2-nitrobenzoic acid). HSA changes were monitored using native PAG electrophoresis and fluorescence spectroscopy. For FA/HSA molar ratios screening, qTLC and GC were used. FAs increase thiol group carbonylation levels from 8% to 20%. The k' values obtained for FAs-free HSA-SH and FAs-free MG-HSA-SH are almost equal (7.5×10(-3) and 7.7×10(-3)s(-1), resp.). Binding of all FAs amplify the reactivity (k' values from 14.6×10(-3) to 26.0×10(-3)s(-1)) of HSA-SH group for 2-3.5times in the order: palmitic, docosahexaenoic, fish oil extract, stearic, oleic, myristic and eicosapentaenoic acid, due to HSA conformational changes. FAs-bound MG-HSA-SH samples follow that pattern, but their k' values (from 9.8×10(-3) to 14.3×10(-3)s(-1)) were lower compared to unmodified HSA due to additional conformation changes of HSA molecules during carbonylation. Carbonylation level and reactivity of Cys34 thiol group of unmodified and carbonylated HSA depend on type of FAs bound to HSA, which implies the possibility for modulation of -SH reactivity (scavenger capacity and antioxidant property) by FAs as a supplement.

The efficiency of compounds with α-amino-ß-mercapto-ethane group in protection of human serum albumin carbonylation and cross-linking with methylglyoxal.

α-Oxoaldehydes, which are produced in higher quantities in diabetes, uremia, oxidative stress, inflammation and aging, react with the amino, guanidine and thiol groups of proteins and cause the formation of advanced glycated end-products and protein cross-linking. To prevent these reactions, the efficiency of low molecular mass thiols with an α-amino-ß-mercapto-ethane group (Cys, penicillamine and N-acetylcysteine (NAcCys, with a blocked amino group)) as scavengers of methylglyoxal, compared with glutathione (GSH) and the biguanidine derivative metformin, was investigated. The time courses of the reactions of the aforementioned compounds with methylglyoxal were assayed. The reactivity of their thiol and amino groups decreased in the order of Cys > penicillamine > GSH > NAcCys and penicillamine > Cys > GSH, respectively. Human serum albumin (HSA) carbonylation in the absence or presence of methylglyoxal scavengers were monitored by the determination of the amino, guanidine and thiol groups' contents, as well as by spectrofluorimetry, CD and native and SDS PAGE. Cys and penicillamine were highly efficient in the prevention of the carbonylation of the HSA-amino (for 80%) and guanidine (for 84% and 55%, respectively) groups and the formation of fluorescent AGEs. GSH and metformin exhibited medium efficiency (reduction of amino group's carbonylation for 60% and guanidine for about 30%); the least efficient was NAcCys. The presence of Cys, penicillamine and NAcCys led to an almost complete protection of the HSA-thiol group's carbonylation, whereas metformin was inefficient. The efficiency in the prevention of protein cross-linking increased in the order of metformin, NAcCys < GSH < penicillamine < Cys. Thus, the substances with an α-amino-ß-mercapto-ethane group as a pharmacophore exhibit great potential as an efficient methylglyoxal scavengers, and are thus promising compounds for medicinal chemistry. In addition, they protect the HSA-SH group and preserve its antioxidative potential, which is very important for the HSA's function in vivo.

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal.

Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys>GSH>NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 + or - 0.9%; 38.2 + or - 0.7% and 39.0 + or - 0.8% for Cys; 26.5 + or - 0.6%; 26.6 + or - 2.6% and 27.4 + or - 2.5% for GSH; 10.8 + or - 0.9%; and 11.2 + or - 0.7% and 12.2 + or - 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.