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316 Heegaard et al.
parameters extracted. In principle, the quantitative characterization may be
performed using the complex tryptic digest mixture directly. This is the case
if there is a suitable resolution and if the interaction kinetics enable migration
shift experiments because then the exact concentration of receptor molecules
need not be known (c.f. Subheading 6, below).
4.1. Method
Most laboratories will have experience in methods for trypsin digestion of
proteins, compared with (74). A very convenient reagent for S-pyridylethylation
of cysteine residues is 4-vinylpyridine (75). A short outline of trypsin digestion
and test for heparin binding is given here:
1. Protein at >1mg/ml is reduced and S-alkylated/amidated/pyridylated, dialyzed
against water and trypsinized in 0.1 M NH 4 HCO 3 using 1–5% (w/w) sequencing
grade trypsin at 37ºC with gentle stirring.
2. The trypsin cleavage is followed by HPLC or by CE to ensure complete digestion
(typically overnight).
3. The digest is dried down (in a Speed-vac centrifuge) in polypropylene tubes.
4. Re-dissolve in 10–20 L water and subject to CE in 0.1 M phosphate, pH 7.4
(see Note 6) in the absence or presence of heparin.
5. A concentration-dependent anodic displacement/disappearance of tryptic peaks in
the profile indicates heparin-binding activity (see Fig. 3).
6. Reactive peptides are purified for identification by preparative CE (see Note 7),
or peaks are mapped by HPLC-MS and collected purified material is used for
spiking analysis to identify the peaks in the CE-profile.
7. Based on the findings, synthetic peptides can now be made and used to characterize
binding quantitatively (76) (see Fig. 4).
5. Conformation Structure-Function Studies
Few possibilities exist for the simultaneous separation of protein conformers
and performance of binding studies. CE is unique in sometimes being able to
achieve such a separation, and thus, folding parameters such as interconversion
◭
Fig. 3. (Continued)Asterisks mark an interacting component and the lower trace in
each figure shows the behaviour of an RP-HPLC-purified tryptic peptide (T3) corresponding
to amino acid residues 14–38 of the parent SAP. The T3 peptide was identified
by mass spectrometry/amino acid composition analysis (111), and its placement in the
structure of an SAP monomer is indicated in (C) by the dark colour (Adapted with
permission from (17)).