Fig. 5.

Dose-dependent JBH treatment converts larger G_nH_noligos from SeHAS HA to smaller G_nH_n species indicating sequential GlcNAc linkages. Aliquots (0.75 µL) of Wash 3 were incubated at 30°C overnight with 4.0 µL of JBH as a combination of active and heat-inactivated enzyme. Samples had no active JBH (black bars; 0 µL active enzyme; 4.0 µL inactive enzyme) or increasing amounts of active JBH (µL: 0.5, white bars; 1.0, cross-lined bars; 2.0, light gray bars; 3.0, stippled bars; 4.0, dark gray bars), and decreasing amounts of inactive JBH, as described in Methods. Members of the G_0–3H₂ (A) and G_0–3H₃ (B) series were analyzed to monitor decreases or increases in each member as a function of increasing active JBH concentration. Significant differences for G₃H_2,3, assessed by paired Student t-tests, between samples with varying JBH activity vs. inactive JBH, are indicated for two independent experiments; values are the mean intensity ± SEM (n = 8): *P < 0.05; **P < 0.005; ***P < 0.0005; ^#P < 0.00005; ^##P < 0.000005. Since starting values for G_1,2H_2,3 and H₂ and H₃ were zero (denoted by zero-line black rectangles), P values for the samples with 1.0–4.0 µL active JBH were compared to the sample with 0.5 µL, the lowest active JBH dose.