An Improved Assay for the N-Acetyl--glucosamine Reducing Ends of Polysaccharides in the Presence of Proteins

Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength

“…The experimental procedure used is detailed in Astériou et al (2001). Because of the presence of HAase in the samples of the reaction mixture, correction for turbidity was performed as described by Astériou et al (2001).…”

“…However, because of the non-linearity of the kinetics generally observed (Astériou et al, 2001;De Salegui et al, 1967;Bonner and Cantey, 1966;Vercruysse et al, 1999), we have shown (Vincent et al, 2003) that it is much safer to follow the entire time course of the reaction and estimate the initial hydrolysis rate of the reaction catalysed by HAase by measuring the slope of that curve at time zero. Thus, for any set of experimental conditions, it is possible to follow the change of colour as a function of time and to determine the initial reaction rate by fitting the experimental points and determining the slope of the curve at time zero (Vincent et al, 2003).…”

“…Nevertheless, as HAase and HA molecules are present together in the samples, precautions must be taken to avoid the interference of proteins with the HA reducing end assay. A variant of the Reissig method has recently been developed in our laboratory (Astériou et al, 2001) and allows us to distinguish two contributions to the absorbance at 585 nm: the first is turbidity, the second is colour and represents the concentration of the reducing ends, which is directly related to the h(1,4) bond cleaved by HAase. When the HA hydrolysis catalysed by HAase is studied with the variant of the Reissig method, the variation of the HA chain concentration is given as a function of time, and the slope of the tangent to the curve directly represents the reaction rate.…”

Biosynthesis of high molecular weight hyaluronic acid by Streptococcus zooepidemicus using oxygen vector and optimum impeller tip speed

“…HA is present in many body tissues and fluids of higher organisms (synovial fluid, vitreous humor of the eye, umbilical cord) and is one of the main components of the extracellular matrix especially in connective tissues (3,4). With its biological functions and unique physicochemical properties, like high water-holding capacity, muco-adhesion, good viscoelasticity and biocompatibility, HA has been widely used in the areas of drug delivery, ophthalmology, orthopedics, rheumatology and tissue engineering (5).…”

Quantitative continuous assay for hyaluronan synthase

“…The column was eluted with H 2 O at a flow rate of 10 ml/h, and 1-ml fractions were collected. The fractions (samples 159-172) containing sugars were identified using a colorimetric assay for carbohydrates containing an N-acetyl group [35,36]. Mass spec [highresolution electrospray ionization-mass spectrometry (ESI-MS)] run in negative mode yielded two major peaks of m/z 775.2 and 797.2 corresponding to the tetrasaccharide and its mono sodium salt, respectively.…”