Biomedical Engineering Reference
In-Depth Information
concentrations led to signifi cant decreases in AT function,
255
a potential basis for development
of thrombosis in severe diabetics.
256
Mechanistically, short-term interaction of glucose with AT
may only produce temporary effects from the reversible Schiff base complex, while longer glu-
cose incubation will lead to impact from the permanent Amadori product. Interestingly, it has
been demonstrated that heparin's catalysis of AT reactivity can be inhibited by nonenzymatic
AT glycation
in vitro
257
and
in vivo
.
257
These data, combined with the observation that inhibi-
tory effects of glycation on heparin activation of AT could be overcome by incubation with Na
heparin before assay,
257
suggest that Schiff base/Amadori rearrangement products are formed at
a particular residue that interacts closely with heparin. This selective glycation hypothesis was
further strengthened by the fact that incubation of AT with 5 mM glucose for 10 days resulted
in uptake of just 0.6 mol of glycose per mole of protein.
256
Other studies have shown that several
residues on AT can form stable keto-amines.
258
However, even reactions with 0.5 M glucose lead
to only one glucosyl modifi cation per AT,
258
inferring that further glycation is unlikely if the
protein is already glycated at one site. All these fi ndings have given impetus to a novel potential
mode for AT conjugation. Since heparin can affect and is affected by glycation of AT, aldose-
containing heparin molecules may be capable of forming Schiff base or Amadori structures at
particular sites in AT. As an adjunct to this concept, previous studies have revealed that a small
portion of heparin molecules in fact terminate in aldose residues as opposed to a serine glycoside
cap.
207
Thus, simple incubation of ATH under physiological conditions may yield covalent ATH.
However, the prevailing thought in the fi eld of nonenzymatic glycation has been that Schiff base-
Amadori rearrangement of proteins with aldose-terminating polysaccharides is highly improb-
able. Although Amadori adducts have formed between insulin and maltose disaccharide,
259
it
was considered that occurrence of Schiff bases between protein amino groups and polysaccha-
rides is remote given that the aldose-end group represents a minor proportion of all residues in
the polymer, approach of polysaccharide to the protein is much more sterically hindered, and
the terminal aldose is much more likely to be in the hemiacetal (masked aldehyde) form.
260
As
a consequence, spontaneous glycation between polypeptide amines and polysaccharide aldose-
termini remained unexplored.
Chan et al. have conducted intensive experimentation to determine if practical preparation of
ATH is feasible by either Schiff base and Amadori rearrangement or reduction of Schiff bases
between AT and commercial UFH. Given that AT binds to and is activated by heparin through a
high-affi nity pentasaccharide, it would be important to develop a reaction setup, which allows for
AT selection of heparin molecules where this active sequence is located close to the aldose termi-
nus. Since only around 10% of molecules in commercial UFH contain a free aldose
207
and only
about one-third of UFH chains have a pentasaccharide,
207
it was deemed that a 200 molar excess
of UFH relative to AT may be necessary.
141
Trial experiments with various polypeptides and poly-
saccharides showed that glycation of protein by the polysaccharide aldose gave variable yields for
a number of factors.
261
One critical issue was reaction temperature. Previous studies have shown
that incubation of monosaccharide aldose with AT can increase the temperature at which thermal
denaturation of the protein occurs.
262
As a corollary, it might be possible that increased temperature
may be necessary to induce the conformation desired for linkage during aldose interaction with AT.
Indeed, experiments revealed that while yields (in terms of AT) of ATH were
C, produc-
tion improved to 50% at 40°C.
261
An ATH synthetic method was standardized with heating of AT
and UFH in phosphate-buffered saline (PBS) for 14 days, followed by removal of free heparin by
hydrophobic chromatography and further purifi cation by anion exchange to isolate conjugate from
unreacted AT.
141
Analyses have verifi ed that the resultant ATH contained one heparin per AT.
141,206
ATH product was partially digested with protease and heparin-containing peptides sequenced to
determine the heparin linkage location within the AT polypeptide. Analyses determined that in
the vast majority of ATH molecules (87%) the heparin aldose was bonded to the N-terminal amino
group, followed by a minor subpopulation of complexes containing heparin chains linked to the
ε-amino at Lys139.
263
∼
5% at 37
°
