Biomedical Engineering Reference
In-Depth Information
HCl. The resulting solutions were centrifuged at 50,000 g for 1 h at 40
o
C to remove insoluble
particles. The ternary water-gelatin-BSA systems with required composition were prepared
by mixing solutions of each biopolymer at 40
o
C. Both the BSA and the dextran solutions
show Newtonian behavior (at temperatures of 40
o
C and at shear rates up to 30 s
-1
) in the
concentration range from 25 to 30 wt % within which the hydrodynamic volumes of the
different macromolecules already overlap.
2.2.
Dynamic Light Scattering
Determination of
Intensity- size distribution, volume-size distribution of hydrodynamic
radii, (R
H
) and zeta potentials of BSA, gelatin, and their mixtures was performed, using the
Malvern Zetasizer Nano instrument (England), using a rectangular quartz capillary cell.
Concentration of one protein in BSA/gelatin mixtures frequently was kept at 0.25 (w/w). For
each sample the measurement was repeated 3 times. The samples were filtered before
measurement through DISMIC-25cs (cellulose acetate) filters (sizes hole of 0.22 μm for the
binary water-protein and solutions and 0.80 μm for the BSA-gelatin mixtures. Subsequently
the samples were centrifuged for 30 seconds at 4000 g to remove air bubbles, and placed in
the cuvette housing. The detected scattering light intensity was processed by Malvern
Zetasizer Nano software.
2.3. Circular Dichroism Measurements
CD measurements of BSA solution alone and in the presence of gelatin were performed
using a Chiroscan Applied Photophysics instrument. Far-UV CD spectra were measured in 1-
mm cells at 20
o
C and 40
o
C. The solutions were scanned at 50 nm min-1 using 2 s as the time
constant with a sensitivity of 20 mdeg and a step resolution of 0.1. The ellipticity at 222 nm,
õ
222, is assumed to be linearly related to mean helix content,
f
H
, which can be calculated
from the Lifson-Roig-based helix-coil model according to the procedure described before by
Luo and Baldwin [26,27].
2.4. Fluorescence Measurements
Fluorescence emission spectra between 280 and 420 nm were recorded on a RF 5301 PC
Spectrofluorimeter (Shimadzu, Japan) at 25
o
C and 40
o
C with the excitation wavelengths set
to 250, 270, and 290 nm, slit widths of 3 nm for both excitation and emission, and an
integration time of 0.5 s. The experimental errors were approximately 2%.
2.5. Turbidimetry
The cloud points of aqueous BSA solutions and their mixtures with gelatin solutions were
determined by measuring the transmittance of the solutions as a function of temperature for
Search WWH ::
Custom Search