ABSTRACT
Stopped-flow kinetic studies have been performed to determine the kinetic parameters of K(+) binding to the fluorescent cryptand F222 and of Na(+)binding to F221 at pH 8.O. The results clearly indicate that a comparatively stable intermediate is formed before the rate-limiting binding step occurs with a rate constant around 30 s(-1) under the chosen experimental conditions. The conversion of the intermediate to the final cation complex is assigned to the final penetration of the already bound, but still partially solvated cation into the ligand's cavity. The main fluorescence intensity change found upon cation binding is attributed to the second reaction step, and not to the fast, initial binding reaction. The comparatively slow overall binding reaction is interpreted on the bases of a special solvate substitution mechanism which, in principle, can also account for the 1500 times slower binding of Ca 2(+) to F221. With regard to time-resolved analytical Na(+) and K(+) determinations, the response times under the chosen conditions are around 20 ms. Differentiation between Na(+) and Ca(2+), for example, is possible with F221 on the basis of completely different response times.
ABSTRACT
Two newly synthesized cryptands act as sensitive Na(+)- and K(+)-selective indicators for cation concentrations above 20 µM. The fluorescence properties change markedly upon cation binding. In addition, the free ligands exhibit a pronounced sensitivity to pH, which is considerably lower for the cation complexes. Time resolved fluorescence is characterized by a decay time of about 5 ns that is attributed to the diprotonated protolytic state of the uncomplexed ligands. Semiempirical calculations show the systematic influence of the nitrogen lone pairs or the N-H bond on the stability of the system. The cause of the strong fluorescence intensity increase observed upon protonation of the fluorescent cryptands may be attributed to an increase in the S1-T x energy gap as a consequence of bridgehead nitrogen protonation.
ABSTRACT
Two N-acetylglucosaminidases were isolated from bovine kidney with a three step procedure featuring affinity purification on 2-acetamido-1,2,5-trideoxy-1,5-iminoglucitol (2-acetamido-1,2-dideoxynojirimycin, II). The major isoenzyme, Hex A, is an alpha, beta hetero-dimer (57 and 52 kDa) with isoelectric points from pH 5.3 to 6.6 and comprised about 80% of the total activity. Its kinetic properties with respect to discrimination between N-acetylglucosaminide, N-acetylgalactosaminide and the corresponding 6-sulfate ester were similar to human hexosaminidase A. The minor isoenzyme, Hex B, a homodimer, isoelectric points 7.0 to 7.4, was similar to Hex A but was without detectable activity with methylumbelliferyl-N-acetyl-beta-glucosaminide-6-sulfate. Inhibition studies with Hex A were carried out with 2-acetamido-2,5-dideoxy-1,5-imino-D-glucopyranose (2-acetamido-2-deoxynojirimycin, (1), the corresponding 1,5-lactam (III), with II and its N,N-dimethyl derivative, and with 2-acetamido-2-deoxy-D-glucono-1,5-lactone (IV). In comparison with N-acetylglucosamine (Ki 1.9 mM) Hex A was inhibited 10(6)-fold better by I, 2600-fold better by II, 2900-fold better by III, and 55,000-fold better by IV. A slow approach to the inhibition equilibrium was observed with I and IV. For IV and Hex A it is the first example of a slow inhibition of a glycoside hydrolase by the corresponding glycono-1,5-lactone. The pH-dependence of Ki for the permanently cationic N,N-dimethyl II (15.4 microM (pH 3.5) to 0.47 microM (pH 7.0)) indicated that formation of the enzyme inhibitor complex is governed by deprotonation of a group with pKa 5.0. The results are discussed with respect to structural features and water accessibility of the active site.
