Quantitative continuous assay for hyaluronan synthase

Abstract

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.

Fig. 1

Schematic representation of the three-enzyme coupled signal decrease assay for PmHAS. PmHAS transfers GlcNAc and GlcUA from UDP-GlcNAc and UDP-GlcUA to the nonreducing end of HA so as to extend the HA chain. The assay relies on coupling the release of UDP produced from Eq. (1) to PEP in the presence of PK (Eq. (2)). Then pyruvate, a by-product of Eq. (2), is reduced to l-lactate with concomitant oxidation of NADH into NAD⁺ (Eq. (3)). The decrease in NADH absorbance is monitored in a continuous fashion.

Fig. 2

Detection limit for the signal decrease assay for PmHAS using purified recombinant PmHAS^1–703. Data were obtained at near-saturating conditions (4K_{M, app}) of UDP-GlcUA, UDP-GlcNAc, and HA₄. Absolute values of the rates are plotted. The standard deviation of repeated values is less than 10%.

Fig. 3

Structure of the Xuorescent HA primer HA₄-ANDA.

Fig. 4

Fluorophore-assisted carbohydrate electrophoresis. Lane 1 contains 3 nmol of HA₄-ANDA and 30 nmol of both UDP-GlcNAc and UDP-GlcUA. Lane 2 contains 3 nmol of HA₄-ANDA. Lane 3 contains 3 nmol of HA₄-ANDA and 30 nmol of both UDP-GlcNAc and UDP-GlcUA in the presence of non-induced cell lysate. Lane 4 shows the extension of HA₄-ANDA on the addition of 3 nmol of HA₄-ANDA combined with 30 nmol of both UDP-GlcNAc and UDP-GlcUA in the presence of induced cell lysate. Lane 5 is a repeat of lane 4 except that the elongated HA₄-ANDA fragments were not treated with acid or boiled prior to loading on a gel (see Materials and methods). The elongated HA₄-ANDA fragment shown in lane 4 was digested with either Streptomyces (lane 6) or bovine testicular HAase (lane 7).

Fig. 5

Representative Michaelis–Menten plot of rate (abs/min) versus [HA₄]. Data were obtained at near-saturating substrate concentrations (≥4K_{M, app}) for UDP-GlcUA and UDP-GlcNAc using the standard assay protocol. Absolute values of the rates are plotted.

Fig. 6

Representative inhibition plot for UDP-Glc obtained at 0.5K_{M, app} for each UDP-GlcUA, UDP-GlcNAc, and HA₄. Data were obtained using the standard assay protocol.

Fig. 7

Representative Dixon plot of 1/rate versus [UDP-Glc]. Data were obtained at 0.5K_{M, app} for each UDP-GlcUA and HA₄ while working at near-saturating conditions for UDP-GlcNAc (4K_{M, app}). Absolute values of the rates are plotted.