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Updated: Jun 27, 2026

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry
Published on: July 20, 2016
Disulfide and thioether linked cytochrome c-oligoarginine conjugates in HeLa cells
Maureen P Barnes1, Wei-Chiang Shen
1University of Southern California, School of Pharmacy, Department of Pharmacology and Pharmaceutical Sciences, Los Angeles, CA 90033, United States.
Abstract:
The intracellular processing and the apoptotic activity of conjugates of oligoarginine and cytochrome c (Cyt c) were studied. Disulfide and thioether linked conjugates were prepared by coupling Cyt c to cysteinyl-nonaarginine, C(R)(9), through SPDP and SMPB cross-linkers, respectively. Internalization of the radiolabeled conjugates was measured, and biological activity via induction of apoptosis was determined using the annexin V and the acridine orange assays in HeLa cells. The internalization of both conjugates is increased when compared to that of Cyt c alone. However, the biological activity of the internalized Cyt c, indicated by apoptosis in HeLa cells, was expressed only in the thioether (SMPB) conjugate, but not the disulfide (SPDP) conjugate or free Cyt c. The addition of the proteasomal inhibitor MG132 increased the apoptotic activity of both the disulfide conjugate and free Cyt c, but not the thioether conjugate. Our results suggest that the intracellular cleavage of the linker in cell penetrating peptide conjugates is critical in determining the fate and activity of biologically degradable cargo molecules.
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