Biomedical Engineering Reference
In-Depth Information
7.2.4
Collagen renaturation
-
-
Before discussing the renaturation of degraded collagen
it is necessary to
consider the essential features of the renaturation of soluble collagen. If the essential
subunit, tropocollagen, is suf
gelatin
ciently denatured by heating, the three strands separate
partially (since there may be some covalent cross-links) or completely to give the
flexible
polypeptide form. Tropocollagen itself is a rod-like particle of M
w
~ 3.45 × 10
5
g mol
−
1
,
so that each strand has M
w
~ 110 000 g mol
−
1
. The length is around 300 nm and the
diameter is ~1.4 nm. The temperature of denaturation depends upon the source (and thus
the exact polypeptide composition), as mentioned above. However, the helix
coil
transition is sharp, and the initial nucleation of the re-formed helix occurs rapidly.
Such a solution is essentially a very ideal form of gelatin, and renaturation studies
show a number of important features.
The kinetics of renaturation can be followed by, for example, optical rotation
measurements, and this approach has been carried out by a number of workers, so
here we only summarize the results. At low concentrations c, the kinetics is
-
rst-order
with respect to c, increasing to second-order at higher concentrations. This is consistent
with a change from intramolecular helix formation at lower concentrations to inter-
molecular helices at higher concentrations. Both nucleation and helix growth are slow;
the nucleation presumably involves just two chains (since three-body collisions are
very unlikely in dilute solutions). The nuclei have a critical size, below which helix
propagation does not occur, and this has been examined by employing special samples
with a very low number of residues. Estimates of the critical size lie in the range 20
-
40,
and indeed a 36-residue peptide can renature, so the lower limit is probably realistic.
Also, for such a 36-residue system, intramolecular folding is very unlikely, so the helix
is formed of 3 × 36-residue strands. Helix growth is slow, but the subsequent propaga-
tion is even slower as the coil-to-triple helix
rate is limited by the presence of
cis-proline residues in the backbone. The subsequent reversion of these to the trans
form allows the helix to extend only gradually so that the overall helix propagation rate
is typically four to six orders of magnitude slower than for double-helical systems such
as carrageenan (
Chapter 5
).
'
zipping
'
7.2.5
From collagen to gelatin
7.2.5.1
Molecular mass distribution
To extract gelatin from collagen, the entire structure of the
fibres has to be disrupted.
Both chemical and thermal treatments are required during the extraction process to
break down both inter-chain covalent and hydrogen bonds. There are two processes of
extraction: one uses alkali and the other acid. Generally, gelatin from bovine bones and
hides is extracted with alkali and gelatin from skins (porcine,
fish) with acid. The
process modi
es the isoelectric point of the chains, which is around pH= 5 for the basic
extract and around pH= 8
9 for the acidic extract (unchanged from native collagen).
The process produces gelatins with various molecular masses. Although initially a
tropocollagen single strand has the known molecular mass, during extraction both
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