2-aminobenzamide labelling

Kotani, N. and Takasaki, S., Analysis of 2-aminobenzamide-labeled oligosaccharides by high-pH anion-exchange chromatography with fluorometric detection, Anal. Biochem., 264, 66, 1998. 

Table 4.5.6 Running conditions for high-performance liquid chromatography analysis of 2-aminobenzamide-labelled oligosaccharides...

M. Wuhrer, C.A.M. Koeleman, C.H. Hokke, A.M. Deelder, Nano-scale LC-MS of 2-aminobenzamide-labeled oligosaccharides at low femtomole sensitivity, Int. J. Mass Spectrom., 232 (2004) 51. 

Merry, A. H. Neville, D. C. A. Royle, L. Matthews, B. Harvey, D. J. Dwek, R. A. Rudd, P. M. Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis. Anal. Biochem. 

G. Brix, A. Schlicker, W. Mier, P. Peschke, M.E. Bellemann, Biodistribution and pharmacokinetics of the F-19-labeled radiosensitizer 3-aminobenzamide Assessment by F-19 MR imaging, Magn. Reson. Imaging 23 (2005) 967-976. 

Fig. 4.5.8 Examples for structures and molecular masses of 2-aminobenzamide (2-AB)-labelled oligosaccharide moieties derived from serum transferrin. Values below the oligosaccharide structures indicate the expected masses (in Da) by matrix-assisted laser desorption ionisation - time of flight analysis.

Various other derivatization strategies have been reported, e.g., V-(2-diethylamino)ethyl-4-aminobenzamide [7], 2-aminonaphthalene trisulfone (ANTS) [60], 2-amino-5-bromopyridine [61]. With the latter label, isotope tagging of the oligosaccharides is achieved, enabhng easy access to sequence information through the diagnostic twin peaks. 

Labeling of the released glycans. To facilitate their detection in the subsequent procedures, the released ohgosaccharides with free reducing ends are labeled. Two methods that are commonly used to label oligosaccharides are reductive amination with a fluorescent compound, such as 2-aminobenzamide and reduction with alkaline NaB H4. 

The main acceptor for ADP-ribose in the in vitro labeling experiments was a protein of mol. wt. of 115—125 kD (Fig. 2). It was assumed to be poly(ADP-ribose) synthetase, since this enzyme has been found to catalyze self-modification reactions [18]. This assumption was verified by incubating nuclear matrices isolated from unlabeled cells with labeled NAD. Figure 4 shows that the tracer was indeed incorporated into acid-insoluble material in a time-dependent reaction. The fact that a specific inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, almost completely inhibited the reaction, indicated that the reaction was catalyzed by this enzyme. This conclusion was also supported by electrophoretic analysis of the labeled products formed (not shown). 

At 2.5 mmolar concentration, thymidine, nicotinamide and 3-aminobenzamide were efficient inhibitors of the enzymatic activity, the apparent Ki with these inhibitors were 55 pM, 139 pM and 23 pM respectively in the case of plasmacytoma. When free mRNPs were incubated with snake venom phosphodiesterase or poly(ADP-ribose) glycohydrolase, but not with RNAses, a release of the labelled ADP-ribose in the incubation medium occurred (25-28). 

Poly(ADP-ribose) was purified almost to homogeneity fi om bull testes by the method of Agemori et al. (5). Purified polymerase was incubated briefly with [ P]NAD (10°C, 30 sec, 5 xM NAD) in the presence of 10 pg/ml Hae IH-digested calf thymus DNA. Incorporation was stopped by the addition of 3-aminobenzamide and unincorporated labeled NAD was removed. Pulse-labeled polymer was originally attached to the enzyme. After alkali treatment it showed a heterogeneous distribution of short chains on a 20% polyacrylamide gel (6). Venom phosphodiesterase digested these to p2p]pR AMP and [ P]AMP, of which AMP represented 4.6% of the counts. After a chase with unlabelled NAD (200 pM NAD, 10 min, 25 °C),... 

Fig. 2. Irreversible modification of the polymerase by dADP-ribose. Ten pi portions of 10% washed HeLa cell lysate were incubated with 10 pi of 25 pM [3 P]NAD (63 G/mmol) and 100 pg/ml DNase I. 5 mM 3-aminobenzamide was included in sample (a). After incubation at 37 C for 5 min, 10% TCA was added to samples a and b. Samples c-e were chilled on ice for 5 min, and centrifuged. The pellets were resuspended in lysis buffer (40 mM Tris-Cl (pH 8), 10 mM Mg(O.CO.CH3)2, 5% Dextran, 0.05% Trition X-1(X) and 0.1 mM CaCl2) plus DNase with (c) no nucleotide, (d) 5 mM unlabeled NAD or (e) 5 mM unlabeled dNAD, and were incubated again for 10 min. The incubation was stopped by the addition of TCA. The precipitates were dissolved in 40 pi of gel buffer containing 1% SDS, 1% 2-mercaptoethanol and were analyzed by 7%-15% polyacrylamide gel electrophoresis. Track (c) controls for losses in the wash. The bands labeled in the presence of 3-aminobenzamide are probably the result of non-enzymatic addition of p PJdADP-ribose that was present in the precursor.

In comparison with polymer synthesis, deoxyADP-ribosylation of polymerase was very slow, but it was DNA-dependent and was inhibited by 3-aminobenzamide, showing that some of the normal polymerase functions were employed. The dADP-ribose seemed to be added to the "automodification domain" (8), since the 67K fragment formed by partial digestion with chymotrypsin was labeled (3). The incorporated dADP-ribose was not removed by poly(ADP-ribose) glycohydrolase (9) showing that the residue was attached directly to the enzyme and was not part of a chain. Also the dADP-ribose residue was attached by an alkali-resistant bond whereas most poly(ADP-ribose) chains are readily removed by alkali. As shown in Fig. 2, neither excess unlabeled NAD nor excess u abeled dNAD would release such [ P]dADP-ribose from the enzyme. This irreversible modification caused inactivation. 

NAD turnover studies. Pulse-chase experiments with [i C]-nicotinamide show that half of the labelled NAD in mature lymphocytes disappears during 8-12 hr of in vitro culture (Fig. 1). If 3-aminobenzamide is added, more than 50% of the radioactivity persists beyond 24 hr. These results indicate that most of die NAD turnover in non-dividing human lymphocytes is due to ADP-ribosylation reactions. Exposure of the cells to 10 xM dAdo causes a marked increase in the turnover of NAD (Fig. 1). The enhanced NAD consumption is associated with a 50% fall in cellular NAD pools after 24 hr (10). Thus, an increase in NAD utilization by lymphocytes for ADP-ribosylation, in response to accumulating DNA strand breaks, caimot be matched by a compensatory increase in NAD synthesis, and the NAD pool becomes depleted. 

Bigge, j. C. Patel, T. P. Bruce, J. A. Goulding, P. N. Charles, S. M. Parekh, R. B. Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal. Biochem. 1995, 230, 229-238. 

Wuhrer, M. Deelder, A. M. Negative-mode MALDI-TOF/TOF-MS of oligosaccharides labeled with 2-aminobenzamide. Anal. Chem. 2005, 77, 6954-6959. 

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