ABSTRACT
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.
Subject(s)
Insulin/metabolism , Peptide Fragments/metabolism , Peptides/chemistry , Serum Albumin, Human/metabolism , Binding Sites/genetics , Circular Dichroism , Fluorescence , Humans , Insulin/chemistry , Insulin/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptides/genetics , Protein Aggregation, Pathological/drug therapy , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/metabolism , Protein Binding/genetics , Protein Structure, Secondary , Serum Albumin, Human/chemistry , Serum Albumin, Human/geneticsABSTRACT
Hyperquenched glassy water (HGW) has been suggested as the best model for liquid water, to be used in low-temperature studies of indirect radiation effects on dissolved biomolecules (Bednarek et al. J. Am. Chem. Soc. 1996, 118, 9387). In the present work, these effects are examined by X-band electron spin resonance spectroscopy (ESR) in gamma-irradiated HGW matrix containing 2'-deoxyguanosine-5'-monophosphate. Analysis of the complex ESR spectra indicates that, in addition to OH(*) and HO2(*) radicals generated by water radiolysis, three species are trapped at 77 K:(i) G(C8)H(*) radical, the H-adduct to the double bond at C8; (ii) G(- *) radical anion, the product of electron scavenging by the aromatic ring of the base; and (iii) dR(-H)(*) radicals formed by H abstraction from the sugar moiety, predominantly at the C'5 position. We discuss the yields of the radicals, their thermal stability and transformations, as well as the effect of photobleaching. This study confirms our earlier suggestion that in HGW the H atom addition/abstraction products are created at 77 K in competition with HO2(*) radicals, in a concerted process following ionization of water molecule at L-type defect sites of the H-bonded matrix. The lack of OH(*) reactivity toward the solute suggests that the H-bonded structure in HGW is much more effective in recombining OH(*) radicals than that of aqueous glasses obtained from highly concentrated electrolyte solutions. Furthermore, complementary experiments for the neat matrix have provided evidence that HO2(*) radicals are not the product of H atom reaction with molecular oxygen, possibly generated by ultrasounds used in the process of sample preparation.
