Biology Reference
In-Depth Information
example, an automated flow analyzer (Burkard Scientific, Uxbridge, UK) based
on the method of Grant
(12)
or a microtiter plate method
(13)
, employing the
same chemistry, can also be used for rapid determination of multiple samples.
13.
The collagen crosslinks represent about 1 mol per mole of collagen, consequently
locating these novel amino acids among the excess of normal amino acids has
historically proved difficult. Prefractionation is therefore carried out to enhance
the relative proportion of the crosslinking amino acids. The preferred method
used in this laboratory is fibrous cellulose although several other methods have
been reported with varying success, Sell and Monnier,
(14)
; Dyer et al.
(15)
;
Takahashi et al.
(16)
; Avery
(17)
.
14.
It is important to record the date of the slurry production as prolonged storage,
e.g., longer than 2 mo, gives rise to an aggregated product that is unusable and
must be discarded. On long-term storage, the 4:1:1 eluant also tends to separate
into two layers and must not be used once this has occurred as the two layers will
not remix into a single phase.
15.
Rehydration in this fashion prevents the formation of an “oily” residue that occa-
sionally occurs if the samples are hydrated with the 4:1:1 eluant directly.
16.
This is best done in a centrifugal evaporator as the sample is then maintained as a
small volume at the bottom of the tube. However, if an evaporator is not avail-
able, then the sample can be freeze-dried in the following manner. The vessel
containing the column effluent is capped and a small hole pierced in the lid. The
vessel should then be frozen at an angle of 45
C freezer. This mini-
mizes the chance of sample loss resulting from its rising up the container during
the freeze-drying process.
17. The technique is based on the use of an automatic amino acid analyzer; we use an
AlphaPlus II (Pharmacia) as previously described
(20)
, but the technique can be
applied to other amino acid analyzers. The supplier of such equipment obviously
provides instructions on its use, so detailed explanations will not be provided
here except for information specific to the analysis of crosslinks.
18. This can be prepared by dilution of Pharmacia's 1.2
M
sodium citrate buffer
pH 6.45 to a molarity of 0.4
M
with water containing 0.1% phenol followed by
adjustment to pH 5.25 with concentrated hydrochloric acid.
°
in a -80
°
19.
Our laboratory uses the AI-450 data handling software from Dionex UK Ltd.
(Camberley, Surrey, UK) (their current version is called 'PeakNet'), to collect
and manipulate the data generated by the amino acid analyzer, although any chro-
matography data-handling software would do. A simple strip-chart recorder
linked to the analyzer would suffice, however, integration of the peak areas would
then have to be performed by manual measurement of the peaks or by cutting out
and weighing the peaks.
20.
The crosslink peaks should be identified by comparison with authenticated
crosslink standards and expressed as moles of crosslink per mole of collagen or
as the reciprocal of this value, i.e., 1 crosslink molecule every “
” molecules of
collagen. The elastin crosslinks Desmosine and iso-Desmosine should be deter-
mined as nmols of crosslink per mg of tissue. The following equation is used
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