Srinivasa K. Rao

A versatile assay for gelatinases using succinylated gelatin.

A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.

REDOX POTENTIAL MEASUREMENTS OF PLASMA IN PATIENTS UNDERGOING CORONARY ARTERY BYPASS GRAFT AND ITS CLINICAL SIGNIFICANCE

The apparent redox potentials (Em) of plasma as a marker of oxidant injury during coronary artery bypass graft (CABG) is determined, and their clinical significance is discussed. We measured plasma Em of normal volunteers (n = 20) and samples drawn at different time points from patients undergoing elective CABG (n = 60) directly and by adding 5 microl (20 mM) oxidants or reductants with known redox potential to plasma (95 microl), using a micro Pt/AgCl combination redox electrode. The Em value stays elevated up to 30 min during the surgery, after the administration of protamine it came down toward a more reduced state. Similar changes are seen with the lactate pyruvate ratio. Smaller changes of Em than normal are observed in plasma samples from patients treated with Aprotinin (antiprotease), Carmeda (heparin-coated) circuit and aspirin reflecting their protective effect. Redox potential (Em) measurements appear to be effective and useful in monitoring redox shifts wherever oxidative stress needs to be monitored.