Table 1.
Functionality determination for Cyt c glycoconjugates
| Protein formulation | Glycosylation degree # | Residual activity $ (%) | Caspase 3 activation % (%) | Caspase 9 activation % (%) | Degradation rate, K d & (x 10 -2 min -1 ) |
|---|---|---|---|---|---|
| Cyt c |
N/A |
100 |
100 |
100 |
10.3 ± 0.1 |
| Lac4-Cyt c |
4.4 ± 0.4 |
94 ± 4 |
95 ± 1 |
95 ± 9 |
9.9 ± 0.2 |
| Lac9-Cyt c |
9.3 ± 0.2 |
97 ± 2 |
86 ± 3 |
93 ± 8 |
9.6 ± 0.1 |
| Dex3(1 kD)-Cyt c |
3.4 ± 0.9 |
98 ± 1 |
91 ± 2 |
96 ± 4 |
9.8 ± 0.2 |
| Dex5(10kD)-Cyt c |
5.2 ± 0.8 |
93 ± 4 |
89 ± 1 |
94 ± 6 |
9.5 ± 0.1 |
| Dex8(10kD)-Cyt c | 8.3 ± 0.4 | 95 ± 3 | 85 ± 2 | 92 ± 7 | 9.9 ± 0.3 |
#Glycosylation degree refers to the number of glycan modified Cyt c lysine residues as determined by 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay. $The residual activity was calculated with respect to the specific activity of native Cyt c.%The caspase 3 and caspase 9 activation is with respect to the activation induced by non-modified Cyt c. The substrates used for caspase-3 and caspase-9 activation assay were 10 mM DEVD-pNA and 4 mM LEHD-pNA, respectively. &The rate of degradation was calculated for Cyt c and Cyt c glycoconjugates after exposure to 1.5 mM H2O2. Each experiment was performed in triplicate, the values averaged, and the ± values are the calculated SD.