Fig. 5. Italian and "Italian-like" amino acid side-chain structures. The Italian amino acid substitution replaces the γ-carboxylate group of E22 (above the dotted magenta line) with an ethylamine group. The "Italian-like" substitution at D23 is produced by substitution with ornithine (Orn), which replaces the β-carboxylate group with an ethylamine group.

Fig. 6. Limited trypsin proteolysis of Dutch, Flemish, Iowa, Italian, Asp23Orn, and Asp23Gly Aβ(21-30) peptides. The peptides were dissolved in 25 mM ammonium acetate buffer at a final pH of 7.4. Trypsin digestions were carried out at 25°C using an E:S ratio of 1:100. All digestions were allowed to proceed for 2 d. The column chemistry used in the LC/MS procedure was diphenyl. Mass spectra are shown of aliquots removed at the indicated times. The identities of the ions are noted above the arrows. Asterisks denote peaks of singly-charged, multiply-substituted sodium adducts separated by 22 amu. (a) Dutch (Glu22Gln) incubated at a concentration of 0.61 µg/µl; (b) Flemish (Ala21Gly) incubated at a concentration of 0.43 µg/µl; (c) Iowa (Asp23Asn) incubated at a concentration of 0.53 µg/µl; (d) Italian (Glu22Lys) incubated at a concentration of 0.40 µg/µl; (e) Asp23Orn incubated at a concentration of 0.43 µg/µl; and(f) Asp23Gly incubated at a concentration of 0.43 µg/µl.

Fig. 7. 2D ¹H ROESY NMR spectra. (a) Expanded regions from selected 2D ROESY spectra of wild type and mutant Aβ(21-30) peptides, as indicated, in 25 mM acetate, pH 6.0, H2O:D2O/9:1, collected at 10ºC with a mixing time of 300 msec. Connectivities are illustrated for sequential Ser-26αH-Asn-27HN(i,i+1) and Ser-26βH-Asn-27HN(i,i+1) resonances. (b) Connectivities are illustrated for sequential Asn-27αH-Lys-28HN(i,i+1) and Asn-27βH-Lys-28HN(i,i+1) resonances.

Fig. 8. Time-dependence of trypsin activity. Enzyme esterase activity was assayed using the chromogenic substrate N_α -benzoyl-L-arginine ethyl ester hydrochloride (BAEE). To do so, BAEE (85 ng/µL) and each trypsin sample (0.85 ng/µL) were mixed in a total volume of 1 ml in various buffers (see below). The mixture was placed in a plastic UV cuvette (BrandTech Scientific, Essex, CT) and A253 data were acquired using a Beckman DU 640 scanning UV/VIS spectrophotometer (Beckman, Fullerton, CA). The "blank" value, which was subtracted from each datum during the reaction process, was obtained by measuring the A253 of the reaction mixture at t = 0. Symbols correspond to trypsin samples incubated in: circles (○), 10 mM sodium phosphate, pH 7.45; squares (□), 10 mM sodium phosphate, pH 6.86; triangles (Δ), 25 mM ammonium acetate, pH 6.86; inverted triangles (Ñ), 25 mM ammonium acetate, pH 6.86, containing Aβ(21-28) (E:S of 1:100); crosses (×), 25 mM ammonium acetate, pH 6.86, containing [D23Orn]Aβ(21-28) (E:S of 1:100). Dashed line is an exponential fit using first-order kinetics. Solid line is the fit using Eq. 10 with κ = 0.77 and γ = 13.7 d^-1.

Fig. 9. Kinetic scheme for trypsin proteolysis of Aβ(21-30). Af, concentration of free Aß peptide; Zf, concentration of free (active) enzyme; C, concentration of Aβ:trypsin complex; P, concentration of products (Aβ(21-28)≡Aβ(29-30)); Zi, concentration of inactive enzyme; K, equilibrium constant for formation of C; first-order (γ) and second-order (α) trypsin inactivation rate constants, respectively; λ, rate constant for peptide proteolysis within C; σ, second-order rate constant for peptide proteolysis. Enzyme release and recycling following proteolysis are not shown.

Fig. 10. Fits of the theoretical progress curves to the experimental data. For ease of viewing, two peptides are shown per panel. Dashed lines are fits using the first-order trypsin autodigestion model, κ = 0 and γ = 18.1 d^-1. Solid lines are fits using the trypsin inactivation kinetics described by κ = 0.77 and γ = 13.7 d^-1. Red squares indicate average experimental digestion levels (see text for details).