Biology Reference
In-Depth Information
to calculate the amount of each collagen crosslink as moles of crosslink/mol
of collagen.
(A
×
RF
(Leu)
×
V
(HCl)
) / (V
(anal)
×
W
(coll)
×
L
×
3.3)
(1)
A
= Area under the crosslink peak (this value is obtained from the data-
handling software or can be determined from a chart recorder connected to the
analyzer by measuring the dimensions of the peak and calculating the sum of
the peak height and the peak width at half the height).
RF
(Leu)
= Response Factor for leucine (obtained from the calibration of the
analyzer using an external leucine standard of known concentration) and is
calculated as follows:
nmols of leucine run on the analyzer/measured area under the leucine peak
(2)
L) of 0.01
N
hydrochloric acid used to dissolve the
sample after CF-1 chromatography.
V
(anal)
= Volume (in mL) of this sample solution run on the amino acid analyzer.
W
(coll)
= Weight of collagen (in
V
(HCl)
= Volume (in
µ
g) contained in the sample applied to the
CF-1 column (this is calculated from the measured hydroxyproline content of
the hydrolyzed sample prior to CF-1).
L
= Ninhydrin Leucine Equivalence value for each crosslink; these are:
HLNL 1.8; HLKNL 1.8; Lys-Pyr 1.7; Hyl-Pyr 1.7; HHL 1.97; I-DES 3.4; DES 3.4.
µ
Worked example:
Let us assume that a sample of hydrolyzed bone was run on a CF-1 column
and that this sample contained 11.4 mg of collagen (
W
(coll)
) obtained from mea-
surement of its hydroxyproline content. After CF-1 chromatography, the aque-
ous eluate was dried and redissolved in 120
µ
L (
V
(HCl)
) of 0.01
M
hydrochloric
acid, of which 60
L (
V
(anal)
) was run on the amino acid analyzer. A peak was
obtained on the analyzer for hydroxylysyl-pyridinoline (Hyl-Pyr) with an area
of 681,731 arbitrary units (
A
), as obtained from the data-handling software
connected to the analyzer. A previous calibration run on the analyzer with a
standard solution of leucine showed that 174,848 units of area were equivalent
to1nmolofleucine. Therefore, using
Eq. 2
,
RF
(Leu)
= 1/174,848 = 5.719
µ
×
10
-6
.
The leucine equivalence value (
L
) for Hyl-Pyr is 1.7.
Therefore, using
Eq. 1
, we have:
10
-6
681,731
×
5.719
×
×
120
= 0.122 mols Hyl-Pyr / mol of collagen
60 x 11.4 x 1.7 x 3.3
21.
Silica-based columns are degraded by prolonged exposure to low pH solvents.
The working life of such columns is very short (months) although they can be
regenerated once or twice by a repacking procedure Avery and Light
(18)
.
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