Aileen E. Boyd

Understanding, improving and using green fluorescent proteins

Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.

Chiral kinetics and dynamics of ketorolac

It has been shown that the analgesic and cyclooxygenase inhibitor activity of ketorolac tromethamine (KT), which is marketed as the racemic mixture of (‐)S and (+)R enantiomers, resides primarily with (‐)S ketorolac and that the ulcerogenic activity of this agent also resides in (‐)S ketorolac. Resolution of individual enantiomers for analysis in plasma samples has been accomplished by two methods: derivatization to form diastereomers that are separated by HPLC, or direct HPLC using a chiral phase column. When mice and rats were given oral solutions of (‐)S and (+)R KT, it was found that the kinetics and interconversion of the enantiomers were species and dose dependent. Interconversion was higher in mice than in rats; when (‐)S KT was administered, 71% of the area under the concentration‐time curve (AUC) was due to (+)R ketorolac in mice, compared with 12% in rats. More interconversion was observed at higher doses; the percent of AUC due to (‐)S ketorolac when (+)R KT was administered increased from 12% to 25% in mice and from 2% to 8% in rats. In general, more interconversion occurred from (‐)S to (+)R ketorolac in the animal studies. Human subjects were given single oral solution doses of racemic KT (30 mg), (‐)S KT (15 mg), and (+)R KT (15 mg). The plasma concentrations of (‐)S ketorolac were lower than (+)R ketorolac at all sample times after racemic KT (22% of the AUC was due to (‐)S ketorolac). When (+)R KT was administered, (‐)S ketorolac was not detectable and interconversion was essentially 0%. When (‐)S KT was administered, significant levels of (+)R ketorolac were detectable and interconversion was 6.5%. After all doses, plasma half‐life was shorter and clearance greater for (‐)S ketorolac than for (+)R ketorolac. Thus, in humans very little or no interconversion of (+)R to (‐)S was observed, and interconversion of (‐)S to (+)R was minimal (6.5%). These data demonstrate that the kinetics and interconversion of the enantiomers of ketorolac is different in animals and humans as well as from most other NSAIDs. This may be due to more rapid excretion or metabolism of (‐)S ketorolac and a different mechanism of interconversion.

Probing the Active Center Gorge of Acetylcholinesterase by Fluorophores Linked to Substituted Cysteines

To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k cat/K m ) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.

An indirect (derivatization) and a direct HPLC method for the determination of the enantiomers of ketorolac in plasma.

An indirect and a direct HPLC method for the quantification of the (R) and (S) enantiomers of ketorolac are described here. The indirect method employs the chiral amine (+)-R-1-(1-naphthyl)ethylamine to form disastereomeric amides; separation of the disastereomeric derivates is achieved by normal-phase HPLC with a mobile phase of ethyl acetate-hexane. The direct method uses a C18 solid-phase extraction column to extract ketorolac enantiomers from plasma; the reconstituted extract is then injected onto an alpha 1-acid glycoprotein chiral column using a mobile phase of isopropanol-phosphate buffer (0.05 M; pH 5.5). Both methods are reproducible, accurate, and stereospecific, and both have equivalent quantification limits (0.02 microgram ml-1 of plasma for each enantiomer), ranges (0.02-2.0 micrograms per aliquot of plasma), precision (% relative standard deviations of < or = 10.5% and < or = 10.8% for (R)- and (S)-ketorolac respectively), and accuracy (mean recoveries of 88.4-110% and 90.1-110% for (R)- and (S)-ketorolac respectively). Results of analyses of clinical samples by the two methods showed excellent agreement (slope near 1.0 and coefficients of correlation between 0.9740 and 0.9864 for both enantiomers).

Nanosecond Dynamics of Acetylcholinesterase Near the Active Center Gorge

To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible ω loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys69-Cys96 ω loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.

Synthesis of fluorescent probes directed to the active site gorge of acetylcholinesterase.

Six organophosphorus compounds linked to fluorophore groups were prepared in an effort to selectively modify the active site of acetylcholinesterase and deliver probes to the gorge region. Two compounds that vary by the length of a methylene (CH2) group, pyrene-SO2NH(CH2)nNHC(O)CH2CH2P(O)(OEt)(F) (where n = 2 or 3) were found to be potent, irreversible inhibitors of recombinant mouse AChE (Ki approximately 10(5) M(-1) min(-1)). Size exclusion chromatography afforded a fluorescently-labeled cholinesterase conjugate.