Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation

The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13–A19 and B12–B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13–A19) and 246 nM (B12–B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13–A19 and B12–B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation

Fullerenol C₆₀(OH)₃₆ Protects the Antioxidant Enzymes in Human Erythrocytes against Oxidative Damage Induced by High-Energy Electrons

Ionizing radiation (IR) can pass through the human body easily, potentially causing severe damage to all biocomponents, which is associated with increasing oxidative stress. IR is employed in radiotherapy; however, in order to increase safety, it is necessary to minimize side effects through the use of radioprotectors. Water-soluble derivatives of fullerene exhibit antiradical and antioxidant properties, and these compounds are regarded as potential candidates for radioprotectors. We examined the ability of fullerenol C60(OH)36 to protect human erythrocytes, including the protection of the erythrocytal antioxidant system against high-energy electrons. Human erythrocytes irradiated with high-energy [6 MeV] electrons were treated with C60(OH)36 (150 µg/mL), incubated and haemolyzed. The radioprotective properties of fullerenol were determined by examining the antioxidant enzymes activity in the hemolysate, the concentration of -SH groups, as well as by determining erythrocyte microviscosity. The irradiation of erythrocytes (650 and 1300 Gy) reduces the number of thiol groups; however, an attenuation of this harmful effect is observed (p 60(OH)36. Although no significant effect of fullerenol was recorded on catalase activity, which was preserved in both control and test samples, a more active protection of other enzymes was evident. An irradiation-induced decrease in the activity of glutathione peroxidase and glutathione reductase became an increase in the activity of those two enzymes in samples irradiated in the presence of C60(OH)36 (p p p 60(OH)36, thus, indicating a lower level of ROS in the system. However, the interaction of fullerenol with the active centre of the enzyme cannot be excluded. We also noticed that radiation caused a dose-dependent decrease in the erythrocyte microviscosity, and the presence of C60(OH)36 reduced this effect (p 60(OH)36 manifested as the protection of the antioxidant enzymes of human erythrocytes against IR-induced damage, which has not been the subject of intense research so far

Interaction of Cationic Protoberberine Alkaloids with Human Serum Albumin. No Spectroscopic Evidence on Binding to Sudlow’s Site 1

Physicochemical studies on drug interactions with human serum albumin (HSA) are relevant for elucidation, at the molecular level, of the processes occurring in vivo. In this work using optical spectroscopic methods (fluorescence, absorption, circular dichroism), we have investigated aqueous HSA solutions containing pharmaceutically important isoquinoline alkaloids, berberine and palmatine. The primary objective was to verify whether the two compounds are located in the subdomain IIA of the secondary HSA structure as reported in literature. We prove that the excited state of Trp214 residue is not quenched by the alkaloids; all observed changes in fluorescence spectra are due to inner filter effects. Furthermore, differential absorption spectra indicate that the ligands remain in a waterlike microenvironment. We infer that bound alkaloid molecules are located at the protein/water interface. Yet, such binding mode can induce some unfolding of the HSA molecule detectable in the far-UV circular dichroism (CD) spectra. We have also performed, for the first time, pulse radiolysis studies of hydrated electron scavenging in the HSA/alkaloid systems and have measured steady-state absorption spectra of irradiated samples. The results reveal that neither berberine nor palmatine is effectively protected by the protein against one-electron reduction, which is consistent with the aforementioned conclusion