Docking results of PEGylated Saks with micro-Plasminogen.

<p>Docking results of PEGylated Saks with micro-Plasminogen.</p

Characterization of the four PEGylated Saks.

<p>(A) SEC analysis was carried out on a Superdex 200 column (1 cm ×30 cm). The column was equilibrated and eluted with 20 mM sodium phosphate buffer (pH 7.2) at a flow rate of 0.5 mL/min. (B) SDS-PAGE analysis of the samples. Lanes 1–6 were standard protein marker, Sak, Sak-mal5k, Sak-ald5k, Sak-mal20k and Sak-ald20k, respectively.</p

SASAs of the PEGylated Saks.

<p>Solvent accessible surface areas (SASA) were calculated by g_sas, a tool in GROMACS package. (A) Total SASAs of PEGylated products. (B) SASAs of the eight amino acids at Sak binding domain.</p

Sedimentation velocity coefficients of the PEGylated Saks.

a<p>The sedimentation coefficient <i>S</i> (10⁻¹³s) in a standard state of water at 20°C.</p>b<p>The ratio of frictional coefficient.</p

Structural characterization of the PEGylated Saks.

<p>Circular dichroism analysis was carried out for the two C-terminally PEGylated Saks (A) and the two N-terminally PEGylated Saks (B). Intrinsic fluorescence analysis was performed for the two C-terminally PEGylated Saks (C) and the two N-terminally PEGylated Saks (D).</p

Schematic presentation of the PEGylated reaction.

<p>(A) PEGylation at N-terminus of Sak. (B) PEGylation at C-terminus of Sak.</p

<i>In vitro</i> bioactivity of the PEGylated Saks.

<p>The <i>in vitro</i> bioactivity was tested by fibrin plate assay. The bioactivities of the PEGylated Saks were compared with that of the unmodified Sak, which was set to 100%.</p

Purification of the PEGylated Sak by ald5k and ald20k.

<p>(A) The sample was loaded on an SP Sepharose HP column (16 mm ×25 mm). The column was equilibrated with 12 ml of 50 mM NaAc–HAc buffer (pH 5.0, Buffer A) and then with 18 ml 0.5 M NaCl in Buffer A at a flow rate of 1.0 ml/min. (B) The fractions corresponding to the proteins were loaded on a Superdex 200 column (2.6 cm ×60 cm) equilibrated and eluted with 20 mM sodium phosphate buffer (pH 7.2) at a flow rate of 3.0 ml/min.</p

Molecular volumes of the PEGylated products.

a<p>V_e: Molecular volumes measured by dynamic light scattering.</p>b<p>V_s: Molecular volumes calculated according to conformations of PEGylated Saks at the end of simulations.</p

RMSDs of the PEGylated Saks during the simulated annealing period.

<p>(A) Sak-mal5k; (B) Sak-ald5k; (C) Sak-mal20k, (D) Sak-ald20k.</p