Biology Reference
In-Depth Information
11.
In addition, pyridoxamine, a commercially available fluorophore not naturally
present in protein hydrolysates, can be added to all samples. Fluorescing at the
same wavelengths as pyridinoline, but migrating earlier, it acts as an internal
fluorescent standard to provide a constant monitor of both column and fluorim-
eter efficiency.
3.8. Pyrrole Crosslink (see Note 23)
1.
Powdered bone or tendon ( see Note 2 ) is placed in a glass universal and repeat-
edly extracted to remove lipids by gentle agitation in acetone at room temperature
until the acetone no longer forms an opaque emulsion when mixed with water.
2.
40-50mg of the bone powder defatted in this way is then suspended in 5 mL of
0.5 M acetic acid, sealed in a tube and left to decalcify for about 2 wk at 4
C. The
acid is then decanted and the sample washed with 4 mL distilled water and then
combined with 400
°
µ
L of TAPSO buffer pH 8.2.
3.
The container should be resealed and the sample denatured by heating in an oven
or dry-block at 110
C for 35 min, following which it is placed in a shaking water
bath and left to equilibrate at 37
°
°
C.
4.
200
L of the trypsin-TPCK solution is added to the sample tube and this digest
mixture is shaken gently for 18 h at 37
µ
°
C.
5.
The digest is then centrifuged for 10 min at 10,000 g to remove particulate material.
6.
40 mL of DAB reagent is added to 200
µ
L of the digest sample and to each of
the calibration standards. Similarly, 50
µ
L of reagent blank is added to further
200
L of digest, to provide a sample blank. After 10 min the samples and blanks
are passed through a 13 mm 0.2
µ
L of each sample, blank, and
calibration standard pipeted into the wells of a 96-well microtiter plate.
µ
m filter and 180
µ
7.
The plate is scanned using a microtiter plate reader set at 570 nm and sample
blank values subtracted from the test sample values. A standard curve is pre-
pared from the calibration standard readings by plotting absorbance against con-
centration of 1-methyl pyrrole and corrected values for the test samples are read
off this curve.
4. Notes
1.
Before undertaking the collagen crosslink analysis of an extracellular matrix,
consideration should be given to the amount of tissue available for the multiple
analytical procedures and the likely collagen content of that tissue.
There exists an approximately 10-fold difference in sensitivity between HPLC
using fluorescence detection and amino acid analysis using ninhydrin detection
in favor of HPLC and, in addition, the HPLC procedure is nondestructive allow-
ing complete recovery of sample for further analysis. However, the intermediate
crosslinks cannot be readily quantified by HPLC without prior derivatization,
with the previously discussed associated problems of multiple peaks for each
component. The advanced glycation end-product pentosidine cannot be quanti-
fied by ion-exchange chromatography as it is retained on the column. Ideally
both analytical procedures should be adopted, but occasionally samples are so
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