Biology Reference
In-Depth Information
3.
After hydrolysis the sample is allowed to cool, the seal is broken and the sample
then brought to between -20
C prior to lyophilization to remove all
residual 6
N
hydrochloric acid (
see
Note 10
).
°
C and -80
°
4.
After drying, the sample can be rehydrated in water (usually 0.5 mL) and divided
according to the requirements of the subsequent analyses (
see
Note 11
).
3.3. Measurement of Hydroxyproline (see Note 12)
1.
A portion of the rehydrated hydrolysate, which is estimated to contain about 16
nmol of hydroxyproline (representing 15
g of collagen), can be analyzed on the
same ion-exchange column as that used for the analysis of the collagen crosslinks
(
see
Subheading 3.6.
), but using a different set of elution buffers.
µ
2.
The column is equilibrated in 0.2
M
sodium citrate buffer pH 2.65 and held at
50
C throughout the run. After the sample has been applied, the column is eluted
with 0.2
M
sodium citrate buffer pH 3.20.
°
3.
Hydroxyproline elutes early from the column (before aspartic acid) and reacts
with ninhydrin to produce a yellow color that can be detected at 440 nm. The
column and detection system are calibrated using an external standard consisting
of a solution of pure hydroxyproline of known concentration.
4.
Collagen is generally considered to contain 14% hydroxyproline by weight, so
the collagen content of the sample can now be calculated from the measured
hydroxyproline value.
3.4. Preparation of a CF-1 Prefractionation Column (see Note 13)
1.
The CF1 cellulose powder (50 g) is first thoroughly wetted with 400 mL distilled
water in a 2-L measuring cylinder, to which is subsequently added 400 mL gla-
cial acetic acid, and finally 1600 mL of butan-1-ol. The resulting 2.5 L of thin
slurry is shaken carefully to ensure complete mixing and suspension of the cellu-
lose and then left to settle (about 20 min) until the bulk of the cellulose is below
the 500 mL mark. The supernatant containing suspended cellulose fines is then
poured to waste leaving approximately 600-800 mL of the 4:1:1 organic mixture
of butan-1-ol:acetic acid:water containing the bulk of the original 50 g of CF-1.
The slurry is now topped up to 1 L with fresh 4:1:1 organic mixture to yield an
approximately 5% CF-1 slurry, which is decanted into a screw-cap container and
stored at room temperature until required (
see
Note 14
).
2.
The prefractionation procedure requires the production of a minicolumn of CF-1.
The top of a 3 mL plastic, Pasteur pipet bulb is cut off and the flow from the tip
reduced, but not blocked, with glass wool or nonabsorbent cotton wool. The CF1
slurry is poured into the pipet through the cut bulb and the cellulose is allowed to
settle, adding more slurry as necessary to produce a settled bed height of 8 cm.
The 3-mL graduation mark on the pipet is a useful guide. Care should be taken to
avoid fluid-filled cavities in the column bed, as this will adversely affect the
chromatographic properties of the column. The newly prepared column should
then be conditioned by passing 2
3 mL of fresh 4:1:1 eluant through the col-
umn. CF-1 columns made in this fashion do not readily dry out, although this
should be guarded against.
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