Particle concentration fluorescence immunoassay of gentamicin.

We report the development of a particle concentration fluorescence immunoassay (PCFIA) for gentamicin using commercially available reagents. The solid phase consists of 0.8 microns polystyrene spheres to which gentamicin antibody is noncovalently bound. Fluorescein-labeled gentamicin serves as the tracer. The assay is carried out on 96-well reaction plates and read using an automated instrument. The assay is accurate over the range of gentamicin concentrations commonly encountered in clinical practice, with day-to-day coefficients of variation of 3-7%. As little as 0.01 mg/L of gentamicin can be detected when sample predilution is omitted. PCFIA is compared with Abbott TDX fluorescence polarization immunoassay for the measurement of serum gentamicin concentrations in routine clinical specimens.

Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain.

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.