The Kinetic Study of the Fluorescence Quenching of COX-1 with Non-steroidal Anti-inflammatory Drugs

Cyclooxygenase or prostaglandin H2 synthase (COX, PGHS) is the key enzyme that catalyzes the conversion of arachidonic acid and O2 to PGH2, the committed step in prostaglandin biosynthesis. Two isoforms of COX are known: One isoform, COX-1, is constitutively produced in most tissues, and appears to be important in maintainance of normal physiological functions of gastrointestinal protection. The second isoform, COX-2, is induced by inflammatory mediators, and the prostaglandins produced by COX-2 lead to inflammation, fever, pain, and are important for process of parturition.
We have studied the inhibition kinetics of several non-steroidal anti-inflammatory drugs (NSAIDs) binding to apo- and holo-COX-1 by fluorescence quenching of tryptophans of enzyme using the stopped-flow system. Addition of NSAIDs (indomethacin, meclofenamic acid, and piroxicam) to apo-COX-1 results in a rapid fluorescence decrease, followed by a slow time-dependent quenching. The fluorescence quenching of holo-COX-1 by indomethacin and meclofenamic acid also occurs in two stages. The time-dependent binding of indomethacin to the enzyme is slower for holo-COX-1 than for apo-COX-1. However, holo- and apo-COX-1 show similar rate of time-dependent binding for meclofenamic acid. In contrast, piroxicam exhibits time-independent inhibition for holo-COX-1. Apo-COX-1 retains an open space at the heme-binding site, creating a deep active channel. NSAIDs might have different binding modes in the active site requiring a multi-step inhibition. The functional groups of NSAIDs would interact with some amino acid residues of the enzyme and influence the binding with COX-1.
The steady state tryptophan fluorescence is quenched by adding collisional quenchers, CsCl, KI, and acrylamide. The quenching efficieny of the quencher decreases in the order of acrylamide >> KI > CsCl, suggesting that the tryptophan residues in COX-1 are located at a hydrophobic environment. More than 80 % of the fluorescence was quenched by acrylamide, indicating that most tryptophan residues are not buried in the interior of the protein. When apo-COX-1 was pre-treated with diclofenac, the collisional quenching becomes less effective. It suggests that binding of diclofenac in the active site may block some tryptophan residues against solvent accessibility.
We have also studied the fluorescence resonance energy transfer analysis of TNS (6-p-toluidinylnaphthalene-2-sulfonate) interacting with apo-COX-1. By measuring the fluorescence decrease of apo-COX-1 and enhancement of TNS emission, it was concluded that there was only one binding site for TNS within apo-COX-1. TNS might bind to apo-COX-1 competitively with NSAIDs. TNS could be used as a probe to determine the inhibition behavior of binding NSAIDs to COX-1.