title: Chemical glycosylation of cytochrome c improves physical and chemical protein stability.
abstract: BACKGROUND
  Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.
  
  
  RESULTS
  Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.
  
  
  CONCLUSIONS
  The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.
authors.forename: Yamixa
authors.initials: Y
authors.lastname: Delgado
authors.forename: Moraima
authors.initials: M
authors.lastname: Morales-Cruz
authors.forename: José
authors.initials: J
authors.lastname: Hernández-Román
authors.forename: Yashira
authors.initials: Y
authors.lastname: Martínez
authors.affiliation: Department of Biology, University of Puerto Rico, Río Piedras Campus, P,O, Box 70377, San Juan, Puerto Rico 00931-3346, USA. kai.griebenow@gmail.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Griebenow
mesh_terms: D017209:Apoptosis; D001682:Biological Availability; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D002474:Cell-Free System; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000493:pharmacokinetics; D003911:Dextrans / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007785:Lactose / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008968:Molecular Conformation; D055550:Protein Stability; D059748:Proteolysis
chemical_list: D003911:Dextrans; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9; D007785:Lactose; D008239:Lysine
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