Millex 0.22 μm pvdf filter

SEC was conducted using an HPLC system (Chromaster; Hitachi High-Technologies Corp.) equipped with an SEC column (Shodex Protein KW-804; Showa Denko K.K., Tokyo, Japan) at an ambient temperature (20-30 °C). The separation range of this column is 30 to ca. 4,000 kDa for proteins. Effluent was monitored using a diode array detector (5430; Hitachi High-Technologies Corp.) with absorbance at 419 nm, which corresponds to λmax of the Soret band of carbonyl Hb. Each modified Hb solution was diluted with PBS to 0.050 mM and was filtered through a syringe-driven filter unit (Millex 0.22 μm PVDF filter; Merck Millipore Ltd., Merck KGaA, U.S.A. or DISMIC-25CS020AS 0.20 μm cellulose acetate filter; Toyo Roshi Kaisha, Ltd., Tokyo, Japan). Then 20 μL of analyte was injected into the SEC column through a sampler. PBS (pH 7.4) was used as an eluent with a 1.0 mL/min flow rate.

For DLS measurements, CM-2, -5, -10, and -20 were reacted with 2.0 molar equivalent DBBF, respectively at initial monomer concentrations, [M]0, of 5.43, 3.10, 3.01, and 1.16 mM to obtain corresponding XLSP-2, -5, -10, and -20 solutions. The Hb concentrations of CMs and XLSPs were adjusted to 0.010 mM in PBS (pH 7.4); then each solution was filtered through a syringe-driven filter unit (Millex 0.22 μm PVDF filter; Merck Millipore Ltd.) to remove dust.
The particle diameter distribution was measured using a nanoparticle size analyzer (SZ-100-S; Horiba Ltd., Tokyo, Japan) equipped with a 10 mW laser (λ = 532 nm) at 25 °C. The lightscattering signal was detected at 173° (backscatter detection). Correlation data were analyzed using software (NextSpec 1.80; Horiba Ltd.). The particle diameter distribution was obtained using a standard monodispersed model based on the scattering light intensity (rather than number or volume), which emphasized the higher molecular weight components. The particle size distribution histograms of CMs and XLSPs are presented in Supporting Information (Figure S1).

The Hb-rPEG particle size distribution was measured using a particle analyzer (ELSZ-2000ZS; Otsuka Electronics Co. Ltd., Osaka, Japan) equipped with a semiconductor laser (λ = 660 nm) at 25 °C. The light-scattering signal was detected at 165° (backscatter detection). The particle sizes of native Hb and purified XL-poly(Hb-PEG) were measured using a nanoparticle size analyzer (SZ-100-S; Horiba, Ltd., Tokyo, Japan) equipped with a 10 mW laser (λ = 532 nm) at 25 °C. The light-scattering signal was detected at 173° (backscatter detection). The Hb concentration of the Hb-rPEG solution was adjusted to 0.10, 0.50, 1.0, 2.0, and 4.0 g/dL (0.015-0.62 mM), and those of native Hb and purified XL-poly(Hb-PEG) solutions were adjusted to 0.73 and 0.12 g/dL (0.11 and 0.019 mM), respectively, in PBS (pH 7.4). Before DLS measurement, each solution was filtered through a syringe-driven filter unit (Millex 0.22 μm PVDF filter; Merck Millipore) to remove dust.