1.Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection.
Cao L1, Wang H, Zhang H. Electrophoresis. 2005 May;26(10):1954-62.
The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited.
2.Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump.
Donnet C1, Caride AJ, Fernández HN, Rossi JP. Biochem J. 1994 Aug 15;302 ( Pt 1):133-40.
Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate.
3.Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.
Jesty J1, Bluestein D. Anal Biochem. 1999 Jul 15;272(1):64-70.
Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate.
4.6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography.
Cao LW1, Wang H, Li JS, Zhang HS. J Chromatogr A. 2005 Jan 21;1063(1-2):143-51.
6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF), a new fluorescein-based amine-reactive fluorescent probe was well designed, synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines in HPLC. It exhibited relatively pH-independent fluorescence (pH 4-9) and excellent photostability. The derivatization was performed at room temperature in 6min. On a C18 column, the derivatives of SAMF with eight aliphatic amines were baseline separated in 28 min with a mobile phase of methanol-water (57:43, v/v) containing 10 mmol l(-1) pH 5.0, H3Cit3-NaOH buffer. With fluorescent detection at lambda(ex)/lambda(em) = 484/516 nm, the detection limit could reach 2-320 fmol (signal-to-noise = 3), which was equivalent to or better than the detection limits obtained from other analytical methods of aliphatic amines. The proposed method has been applied to the determination of the aliphatic amines in environmental and food samples such as lake water, red wine, white wine, and cheese with satisfying recoveries varying from 95 to 106%.
