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. 2022 Jan 10;23(2):719.
doi: 10.3390/ijms23020719.

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability

Yeonje Cho  1 , Armin Mirzapour-Kouhdasht  1   2 , Hyosuk Yun  1 , Jeong Hoon Park  3 , Hye Jung Min  4 , Chul Won Lee  1
Affiliations

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability

Yeonje Cho et al. Int J Mol Sci. .

Abstract

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 - Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.

Keywords: cobalt binding; peptides; stability; structure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of peptide-based metal chelate for cancer diagnosis and therapy.
Figure 2
Figure 2
Change in the UV spectrum of 0.5 mM wAlb12 by Co(II) binding at wavelengths between 230–360 nm.
Figure 3
Figure 3
Change in the UV spectrum of peptides by Co(II) binding. wAlb12 (A) and SMAP18 (G2–G13) (B) at 230–360 nm.
Figure 4
Figure 4
The CD and NMR spectral changes of peptides by Co(II) binding. The CD spectra of wAlb12 (A) and SMAP18 (G2-G13) (B). (C) 1D proton NMR spectral changes of the wAlb12 by adding of CoCl2. Arrows indicates that the peaks were largely broadened out by adding of CoCl2.
Figure 5
Figure 5
The LC-MS analysis and molecular weights of wAlb12 and wAlb12 + Co(II). (A) The HPLC chromatogram of wAlb12 and wAlb12 + Co(II), (B) ESI-MS spectrum of wAlb12, and (C) ESI-MS spectrum of wAlb12 + Co(II).
Figure 6
Figure 6
Measurement of binding affinity of the wAlb12 to Co(II) by isothermal titration calorimetry (ITC). The ITC analysis was performed three times and a representative titration curve was shown.
Figure 7
Figure 7
Stability of wAlb12+Co(II). Proteolytic stability of wAlb12 and wAlb12 + Co(II) complex (A) and human serum stability of wAlb12+Co(II) complex in vitro (B).
Figure 8
Figure 8
The cell viability of the wAlb12 and wAlb12 + Co(II) complex. Cell viability in RAW 264.7 cell (A) and in HEK 293t cell (B).
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