Biology Reference
In-Depth Information
chloride, and 0.005
M
dithiothreitol according to the method of Avery and
Bailey (
11
). The insoluble collagenous network that remains is recovered by
filtration through a 380-
µ
M
copper sieve.
3.
This volume is not critical, although too much could result in under reduction
because of over dilution, and too little could result in the sample being carried
out of the reducing reagent by gaseous hydrogen.
4.
The volume of hydroxide should be as small as possible (
L) to avoid altering the
pH of the sample buffer. The weight of borohydride used for reduction assumes
30% dry matter in the sample and 30% collagen. As a consequence, the propor-
tion of sodium borohydride to collagen will be 1 part to 10 parts of collagen. For
convenience, with multiple reductions, the requisite amount of borohydride for
all samples can be dissolved in a volume of the ice-cold sodium hydroxide and
then appropriate volumes immediately pipeted into each sample as required.
µ
5.
At this point, excess sodium borohydride will rapidly release gaseous hydrogen
with a potential risk of sample loss. This should be kept in mind when selecting
the vessel for the reduction procedure.
6.
If the sample is very small, it is better to carry out all the above procedures in a
vessel suitable for subsequent acid hydrolysis to avoid loss of sample.
7.
The hydrolysis vessel is classically of borosilicate glass and can be reused after
cleaning with chromic acid.
8.
The ratio of sample to volume of acid is not critical provided the sample concen-
tration does not exceed 10 mg/mL when certain resistant peptide bonds may not
be cleaved.
9.
It is customary to perform hydrolysis under a barrier of nitrogen gas (elimination
of oxygen), however, the presence of oxygen is not known to influence collagen
crosslink assays, though certain other amino acids are effected.
10.
C the hydrolysate will not be frozen, however, without prior chilling
there is a considerable risk of sample loss owing to boiling in the reduced pres-
sure of the dryer. The freeze-drying apparatus must be rigorously defended from
attack by hydrochloric acid vapor that will destroy seals, welds, and pump valves
very rapidly. We use a glass vapor trap at -110
Even at -80
°
C to protect the vacuum pump,
which itself is of a specialist design to resist corrosion. We also maintain a strict
monthly vacuum pump oil-change regime.
°
11.
g of collagen is sufficient for measurement of crosslinks by HPLC, but for
ion-exchange 1 mg of collagen or more is required with a CF-1 prefractionation
step. It is important that the CF-1 column is not overloaded, so it is recommended
that no more than 30 mg dry weight of sample be run on a CF1 column.
50
µ
12.
The amino acid hydroxyproline is almost unique to collagen; present in mamma-
lian collagen at about 95 residues per 1000, it is used to determine the total col-
lagen content. This determination is crucial to subsequent procedures because
the final crosslink quantification is expressed as moles of crosslink per mole of
collagen. The most accurate method to use is the ion-exchange column used for
crosslink analysis, but using the standard buffer gradient for amino acid analysis
as described in the text. However, other analytical techniques are available, for
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