Figure 4. HPLC and MALDI-TOF analysis of (GlcNAc)₄ products upon treatment with LytB.

The reaction was carried out at 37 °C in Pi buffer, pH 7.0. Aliquots of the reaction mixture were withdrawn at different incubation times and compounds were detected following the absorbance at 205 nm (arbitrary units). (a) Incubation of (GlcNAc)₄ (84 μM) with LytB (4.2 μM). The substrate and products formed during the reaction are shown at the top. (b) Deconvolution of the elution profile after 30 h of incubation from panel (a). (c) Detection of transglycosilation products by HPLC and MALDI-TOF after 30 h of incubation of (GlcNAc)₄ (1.5 mM) with LytB (4 μM). Product masses (m/z) correspond to the [M + Na]⁺ species of (GlcNAc)₅ and (GlcNAc)₆ together with the hydrolysis products. Monoisotopic masses [M + Na]⁺ are presented for each of the compounds detected. Controls without protein at the longest incubation time are shown (C traces). Peak labels indicate the number of GlcNAc units and the anomeric form (α or β) of each species, assigned by using GlcNAc oligomers (n = 1–6; 300 μM each) as standards (S trace).