Fig 4. Amidolytic activity of nSK/truncated PEG-SK complexes with HPG/μPG at 37°C and 4°C.

Pre-incubated HPG/uPG with nSK/truncated PEG-SK derivatives were added into the cuvette containing chromozyme ^® PL. Amidolytic activity generation were monitored by change in absorbance at A₄₀₅ by hydrolysis of the substrate S-2251. (A) Graph displays amidolytic activation by native SK (black circles) and SK1-383C-PEG20 (green rectangles) in complex with μPG at 37°C. (B) Graph depicts the effect of conjugation of PEG moiety at N-terminal of derivative SK1-383N-PEG10 (red rectangles) by blocking its amidolytic activation in comparison with nSK (black circles), after making complex with μPG, at 37°C. (C) Graph displays the blocking of pathway I by N-terminally PEGylated SK truncated derivatives i.e. SK1-383N-PEG10 (red rectangles), SK1-383N-PEG20 (blue triangles) in comparison with nSK (black circles), when in complex with uPG, at 4°C. (D) Graph depicts no amidolytic activation by SK1-383N-PEG10 (red rectangles) and SK1-383N-PEG20 (blue triangles) in comparison with nSK (black circles), after making 1:1 complex with HPG, at 4°C. Statistical analysis showed significant difference (p-value < 0.0001, n = 6, one-way ANOVA test).