Quantification of chitooligosaccharides by FACE method: Determination of combinatory effects of mouse chitinases.

Highlights • FACE is a simple, qualitative and quantitative method.• The standard curve warrants quantification of chitooligosaccharides of up to 10 nmol regardless on used buffer system.• Our improved FACE method enable us to quantify chitooligosaccharides produced by chitinases at pH 2.0–8.0.• Determination of the combinatory effects of the Chit1 and AMCase using the FACE method.


a b s t r a c t
Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc) 2 , (GlcNAc) 4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0-8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc) 2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing. a r t i c l e i n f o

Method details
Background Fluorophore-assisted carbohydrate electrophoresis (FACE) is a method based on fluorescent labeling of the reducing ends of oligosaccharides. It is a very sensitive technique (pmol levels) as compared to high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectrometry and is often used for detection of very low oligosaccharide quantities [1 , 2] .
ANTS and 2-aminoacridone (AMAC) have been used for fluorescent labeling of the reducing end of oligosaccharides. Jackson (1990) has shown that ANTS can label the reducing end of 35 different oligosaccharides [1] . Recently, we have found that the by-products in the fluoresceinated reaction were formed by labeling at pH > 5 and introduced a pre-acidification step to suppress such products formations [3] . We hypothesized that the improved Jackson method using ANTS could be suitable for chitooligosaccharides labeling. Using this method, we determined the combinatory effects of mouse chitinase in natural chitin substrates processing. Separation and quantification 1. The 40% polyacrylamide gel was prepared according to the composition in Table 1 . 2. The samples were separated by polyacrylamide gel electrophoresis (PAGE), as described previously [1 , 3] .
2. The oligosaccharide was dissolved in water to a concentration of 1 mM. 3. The standard solution was labeled by the method described above.

Result and discussion
As previously reported, the improved FACE method can quantify chitooligosaccharides of various sizes that are directly obtained from enzymatic reactions at pH 2.0-8.0 using several buffer systems commonly employed in the biochemical evaluation of chitinolytic activities [3] . Here, we established a standard curve ( R 2 = 0.998; y = 25.473x + 4.4025) demonstrating the linear association between fluorescence intensity and (GlcNAc) 2 level. The standard curve warrants quantification of chitooligosaccharides of up to 10 nmol regardless on used buffer system ( Fig. 1 ).
The (GlcNAc) 2 production efficiency by Serratia ChiA and ChiB combined was 2-fold higher when compared with a single enzyme showing that the Serratia chitinases act synergistically in chitin degradation [7 , 8] . Chit1 and AMCase mRNA levels in monocytes and macrophages are responding to cytokines. The expression level of Chit1 in activated macrophages is higher than that of AMCase while lower in lipopolysaccharide (LPS)-treated monocytes [9] . To clarify the mutual effects of Chit1 and AMCase on chitin degradation, we analyzed the degradation products from colloidal chitin by their combination at pH 5.0 or 7.0 and incubation at 37 °C for 1 h. The (GlcNAc) 2 production efficiency by the combination of the enzymes was lower than that of the calculated sum (theoretical level) of their respective activities at pH 5.0 ( Figs. 2 A and 3 A) while at pH 7.0, the dimer levels were comparable or slightly lower ( Figs. 2 B and 3 B). These results are consistent with the previously reported data using (GlcNAc) 4 in the condition where large amounts of the substrate remain present [10] . These results suggest that the two enzymes did not compete for the substrates. Our results indicate that, in contrast to the bacterial chitinases, the synergistic effect of mouse enzymes is less pronounced or absent at pH 5.0. At pH 7.0, on Chit1 and AMCase have no synergistic effect and suggest that these enzymes may act independently under various pH conditions [10] .