Abstract
Three regioisomeric radiolabelled spacer-modified oligosaccharides: methyl 4′- O-(4- S-(3-azi-4-α- d-glucopyranosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranosyl]-α-maltoside ( 12a, G1-G3*), methyl 4- O-[4- S-(3-azi-4-α-maltosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranosyl]-α- d-glucopyranoside ( 15a, G2-G2*) and methyl 4- S-(3-azi-4-α-maltotriosyloxy-1-[ 3H]butyl)-6-deoxy-4-thio-α- d- xylo-hex-5-enopyranoside ( 16a, G3-G1*) were synthesised and used as photoaffinity probes for the chemical modification of porcine-pancreatic alpha-amylase (PPA). Incorporation of covalently attached radioactivity amounted to 25–38% of the stoichiometric value. Tryptic digestion of the three labelled protein preparations PPA-G1-G3*, PPA-G2-G2*, and PPA-G3-G1* and the purification of the labelled peptides by fractional HPLC yielded altogether six pure components. On the basis of the published three-dimensional structure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the catalytic site. G1-G3-I and G2-G2-I were part of the surface binding site. The major component derived from PPA, labelled by G3-G1*, corresponded to an area that is neither close to the active site nor to the surface starch-binding domain, which clearly indicates the presence of a third, hitherto undetected, substrate-binding site.
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