Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

The Ω-loop at the active site of β-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of β-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics.

Fluorophore-labeled beta-lactamase as a biosensor for beta-lactam antibiotics: a study of the biosensing process.

The fluorescein-labeled E166C mutant of the PenPC beta-lactamase (E166Cf) represents a successful model in the construction of "switch-on" fluorescent biosensors from nonallosteric proteins (Chan P.-H. et al.

Determination of cadmium in oyster tissue using isotope dilution inductively coupled plasma mass spectrometry: comparison of results obtained in the standard and He/H2 cell modes.

An inductively coupled plasma quadrupole mass spectrometer equipped with an octopole collision/reaction cell was used for the determination of cadmium in oyster tissue samples using isotope dilution inductively coupled plasma mass spectrometry. The oyster samples in question were found to contain Mo and Zr. In our feasibility study on a Cd standard solution (10 microg L(-1)) containing a matrix of Mo (1000 microg L(-1)) or Zr (250 microg L(-1)), the potentially interfering species (MoO(+) or ZrO(+)) present at the analytical mass of cadmium concerned (m/z 111, 112 or 114) was reduced effectively through the use of a mixture of He and H(2) as cell gases.

High-throughput determination of seven trace elements in food samples by inductively coupled plasma-mass spectrometry.

A method was developed for high-throughput determinations of 7 elements in food samples, namely antimony (Sb), arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), and tin (Sn). The samples were digested by closed-vessel microwave-assisted digestion using concentrated nitric acid (HNO3) as the medium, followed by microwave- assisted evaporation to concentrate the sample solutions before dilution to the desired volume. The microwave-assisted evaporation procedure effectively reduced the final acid concentration to around 8% before analysis by inductively coupled plasma-mass spectrometry (ICP-MS).

Fluorescein-labeled beta-lactamase mutant for high-throughput screening of bacterial beta-lactamases against beta-lactam antibiotics.

The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader.

Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase.

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent.