Biomedical Engineering Reference
In-Depth Information
topography of the accessible surface of water soluble complexes of proteins are differ
markedly from the “free” proteins. Therefore it can be assumed that all these factors may
affect the interaction. Most experiments were performed in the absence of low molecular salt
to enable a comparison of the present data with rheological experiments with the single phase
and two phase systems. Because thermal denaturation is usually accompanied by an
aggregation of protein molecules leading to optical changes, the intensity of the transmitted
light was also measured to study the thermal stability of BSA in solutions in the presence of
gelatin. All measurements were performed after previous holding of biopolymer solutions and
their mixtures during 12-15 h. Previous experiments shown that longer store of solutions
before measurement not accompanied by the additional contribution to the data of
measurements. BSA or plasma albumin is a well-known globular protein (M
W
=67 kDa) that
has the tendency to aggregate in macromolecular assemblies. Its three-dimensional structure
is composed of three domains, each one formed by six helices. 17 disulphide bonds are
located in BSA molecule. The most common molecular form is a prolate ellipsoid (4.1 nm x
14.1 nm) [16]. Gelatin is a protein derived by thermal denaturation of collagen. The protein is
unique in that it is made up of triplets of amino acids, gly-X-Y. The X and Y can be any
amino acid but the most common are proline and 4-hydroxyproline, which have a five
membered ring structure. This is most important because it lies at the root of gelatine's unique
ability to form smooth, elastic, thermo-reversible gels [17]. These biopolymers are well
known, widely used in industry for their textural and structuring properties [16,17], and their
capacity to form complexes with polyelectrolytes is known from literature (see for example
[18-21] ).
2.
E
XPERIMENTAL
S
ECTION
2.1. Materials
The BSA Fraction V, pH 5 (Lot A018080301), was obtained from Across Organics
Chemical Co. (protein content = 98-99%; trace analysis, Na < 5000 ppm, CI < 3000 ppm, no
fat acids were detected). The isoelectric point of the protein is about 4.8-5.0, and the radius of
gyration at pH 5.3 is equal to 30.6 Å.[22] The water used for solution preparation was
distilled three times. Most measurements were performed at pH 5.3. The extinction of 1%
BSA solution at 279 nm was
A
1cm 279) 6.70, and that value is very close to the tabulated
value [23] of 6.67. For the molecular weight of BSA, we use 69 kDa (582 amino acid
residues), in accordance with amino acid analysis data.[24]
The gelatin sample used is an ossein gelatin type A 200 Bloom PS 8/30 (Lot 09030)
produced by SBW Biosystems, France. The Bloom number, weight average molecular mass
and the isoelectric point of the sample, as reported by the manufacturer, are, respectively,
207, 99.3 kDa and 8-9. Since the commercial sample contained traces of peptides and various
substances regarded as impurities, an additional purification by washing with deionised water
for 3 h at 5
o
C, was used. The major characteristics of the purified gelatin were described in a
previous paper [25]. To prepare molecularly dispersed gelatin solutions, deionized water was
gradually added to the gelatin, and stirred first at 60
o
C for 20 min and then at 40
o
C for 1 h.
The required pH values of the solutions (5.4) was adjusted by addition of 0.1-0.5 M NaOH or
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