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3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a clinically used anticoagulant drug. Previously, we have developed a method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes and a fourth unidentified protein. The unknown protein degrades adenosine diphosphate (ADP), the by-product of PAPS synthesis reaction. To further understand and control the process of in vitro enzymatic PAPS synthesis, we decide to identify the fourth protein and develop a defined method to synthesize PAPS using purified enzymes. Here, we show that the purified Nudix hydrolase NudJ degrades ADP at high efficiency and serves as the fourth enzyme in PAPS synthesis. Under the defined condition of PAPS synthesis, all of the 10-mM ADP is hydrolyzed to form adenosine monophosphate (AMP) in a 15-min reaction. ADP is a better substrate for NudJ than adenosine triphosphate (ATP). Most importantly, the purified NudJ does not cleave the product PAPS. The removal of ADP makes the PAPS peak more separable from other components in the chromatographic purification process. This developed enzymatic approach of PAPS production will contribute to the chemoenzymatic synthesis of heparin. [ABSTRACT FROM AUTHOR]

The authors describe an electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles (AuNPs) and reduced graphene oxide that was functionalized with hemin (hemin-rGO). Capture DNA (a 21-mer) is immobilized on the AuNPs via gold-thiol chemistry. The detection scheme relies on the measurement of the differential pulse voltammetric current change of the oxidation-reduction reaction of hemin before and after DNA hybridization has occurred. The decrease in the voltammetric currents of iron (III) (best measured at −0.3 V vs. Ag/AgCl) is linearly related to the concentration of complementary DNA. The calibration plot covers the 1.0 aM to 0.1 pM concentration range, and the detection limit is as low as 0.14 aM (at a signal-to-noise ratio of 3). The biosensor was successfully applied to the direct determination of complementary DNA in self-prepared samples by using the standard addition method. [ABSTRACT FROM AUTHOR]

A nanocomposite was prepared that consists of chitosan, reduced graphene oxide and gold nanoparticles by in-situ and simultaneous reduction of graphene oxide and hexachloroaurate whereby chitosan acts as a reducing and stabilizing agent. The nanocomposite was then deposited on a glassy carbon electrode, and glucose oxidase (GOx) was immobilized on its surface to obtain a glucose biosensor. The immobilized GOx displays fast electron transfer with a transfer rate constant of 2.80 s. Operated at a working voltage of −0.45 V (vs. Ag/AgCl), the sensor gives a linear response to glucose in the 0.05 to 1.2 mM concentration range, with a sensitivity of 13.58 μA mM cm, and a 0.52 μM detection limit. The apparent Michaelis-Menten constant is 2.39 mM. It also possesses good selectivity, reproducibility and stability. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]