Table 1.

Table 1a. The pKa Values for SNAFR-1 Based on Absorption and Emission Dataa
method	λex	λisosbestic	λ1/λ2	Rmax	Rmin	pKa
em	325		627/597	1.33	0.54	8.38
em	325		575/597	1.42	0.35	8.32
em	325		541/597	2.31	0.06	8.32
em	488		630/600	1.16	0.51	8.35
em	488		575/600	1.47	0.32	8.36
em	488		542/600	2.47	0.05	8.33
em	514		623/595	1.44	0.57	8.34
em	514		575/595	1.33	0.36	8.32
em	514		539/595	2.16	0.02	8.31
abs		484	543/484	3.76	0.88	8.52
abs		484	511/484	2.04	1.23	8.54
abs		484	469/484	0.48	0.37	8.47
abs		394	380/394	1.41	1.14	8.68
abs		394	344/394	2.73	1.90	8.58
abs		327	380/327	0.52	0.40	8.51
abs		327	344/327	0.99	0.67	8.51
abs		327	312/327	1.17	0.99	8.54
abs		304	312/304	0.91	0.75	8.48
abs		304	296/304	1.38	1.13	8.54
abs		304	265/304	2.79	2.07	8.48
$\text{p}H=\text{p}{K}_{\text{a}}+c\left[log\frac{R-R_{min}}{R_{max}-R}\right]+log\frac{I^{\text{a}}}{I^{\text{b}}}$

Table 1b. The $\text{p}{K}_{\text{a}}^{\ast }$ Values for SNAFR-1 Estimated Using the Forster Equationa
	neutral form		anionic form
methods	ν̃neutral	λneutral	ν̃anion	λanion	ν̃anion − ν̃neutral	$p{K}_{\text{a}-\text{em}}^{\ast }$	$p{K}_{\text{a}-\text{abs}}^{\ast }$
absorption	19 495	512.94	18 451	541.99	1045	6.15	6.34
excitation	19 608	510.00	18 519	540.00	1089	6.05	6.25
emission	18 484	541.00	16 103	621.00	2381	3.34	3.54
0–0 absorption	18 789	532.23	17 248	579.77	1541	5.11	5.30
0–0 excitation	19 084	524.00	17 271	579.00	1813	4.54	4.73
$\text{p}{K}_{\text{a}}^{\ast }=\text{p}{K}_{\text{a}}-\frac{N_{\text{A}}hc({\stackrel{\sim }{\nu }}_{\text{anion}}-{\stackrel{\sim }{\nu }}_{\text{neutral}})}{2.303RT}$

^aThe pK_a of SNAFR-1 in 50 mM phosphate buffer containing 0.25% DMSO was determined using the above equation.^4a The pK_a is taken to be the intercept of the plot of pH versus the first log term in the equation, where c is the slope; R is the ratio from the spectral data at λ₁ and λ₂; R_max and R_min are the limiting values of this ratio; and I^a/I^b is the ratio of the spectral intensity in acid to that in base at the wavelength chosen for the denominator of R. This last term may be neglected by choosing an isosbestic or isoemissive point as the denominator of R.

^aThe $\text{p}{K}_{\text{a}}^{\ast }$ of SNAFR-1 in 50 mM phosphate buffer containing 0.25% DMSO was determined using the above equation^11e with ν̃_anion − ν̃_neutral determined from various methods. $\text{p}{K}_{\text{a}}^{\ast }$ is the pK_a at the first electronically K_a excited state (S1). N_A is the Avogadro’s number. The h is the Plank’s constant. The c is the light speed in the unit of cm/s. The ν̃ _anion and ν̃_neutral (cm^{− 1}) are the optical frequencies of the 0–0 transition in the neutral and anionic form, respectively. The R is the ideal gas constant. The T (K) is the temperature where absorption and fluorescence spectra were collected. The λ (nm) is the wavelength. The $\text{p}{K}_{\text{a}-\text{em}}^{\ast }$ is the pK_a of S1 state calculated using pK_a–em = 8.34. The $\text{p}{K}_{\text{a}-\text{abs}}^{\ast }$ is the pK_a of S1 state calculated using pK_a–abs = 8.53. The 0–0 absorption/excitation is the intersection of the emission with absorption/excitation spectra of neutral or anionic form, respectively.