Table 1.

Rate constants for quinacrine mustard reactions with Cys mutants and with 2-mercaptoethanol

Rate constant	Cys mutant			2-Mercaptoethanol
	T244C	V255C	E262C
k, M−1·s−1	4,930  ± 1,100 (3)	520  ± 150 (3)	146  ± 23 (2)	0.044  ± 0.013 (2)
kMut/k2ME	112,000	11,800	3,320	1

Oocytes expressing mutant receptors were treated alternately with ACh at ≥5× EC₅₀ for 5–10 s and with 5 μM (T244C) or 100 μM (V255C and E262C) quinacrine mustard and ACh (same concentration as in test responses) for 10 s. The oocyte was washed with CFFR for 3–5 min after each application of ACh with and without quinacrine mustard. The equation I = I_o + (I_inf − I_inf)e^−kqt, where q is the quinacrine mustard concentration, was fit to the peak currents of the test responses as a function of the reaction time, and yielded k, the second-order rate constant. Quinacrine mustard (100 μM) in 115 mM NaCl, 10 mM Hepes, and 1 mM EDTA (pH 7.2) was mixed with 2-mercaptoethanol (final concentration 122 μM). The mixture was kept at 23°C under argon, and 0.5-ml aliquots were taken initially and every hour for 6 h. The aliquots were mixed with 400 μM DTNB in 200 mM Tris buffer (pH 8.0). The absorbance at 412 nm was divided by 13,600 to obtain the remaining SH concentration. The SH concentration in the reaction mixture as a function of time was fitted with a second-order kinetic equation to obtain the rate constant. The mean rate constants, the mean error, and the number of determinations are given.