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.
Highlights
There has been a focus on finding effective transporter vehicles for drugs
A derivative of insulin labeled with 4-(1-pyrene)butyric acid Py-Ins (1) was used for the tests, as well as two labeled insulin amyloidogenic cores: Py-LYQLENY-OH (2) analog of A13-A19 and Py-VEALYL-OH (3) derived from the B12-B17 fragment (Figure 1)
We found no increase in fluorescence intensity, which was comparable to the values obtained for Thioflavin T (ThT)
Summary
There has been a focus on finding effective transporter vehicles for drugs. One of the most difficult challenges associated with the design of new DDS is that they should not interact with the immune system [2,3] This requirement is fulfilled by Human Serum Albumin (HSA), the most abundant plasma protein. This protein can reversibly bind various ligands such as lipids, hydrophobic compounds, and metal ions (copper, nickel, and calcium) with high binding affinity, acting as a carrier of various ligands in the circulatory system. This plasma protein has the potential to increase drug retention within tissues [26,27,28]
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