Table 3.

Simulations of possible results of experiments to help assess if Ca was “lost” to parvalbumin

	1	2	3	4	5	6	7
		Case 1: No Mg and no parvalbumin		Case 2: No Mg and with parvalbumin		Case 3: With both Mg and parvalbumin
		Muscle solution	With EGTA	Muscle solution	With EGTA	Muscle solution	With EGTA
(all concentrations in µM)
Rows 1–5 are assumed (typical or expected total values for sample)
1	CaT	21.234	21.234	21.234	21.234	21.234	21.234
2	BAPTAT	120.0	120.0	120.0	120.0	120.0	120.0
3	EGTAT		1,000.0		1,000.0		1,000.0
4	ParvT			7.0	7.0	7.0	7.0
5	MgT					2,000.0	2,000.0
Ca2+ and Mg2+ values adjusted to give CaT and MgT values above
6	Ca2+	0.0472	8.9 × 10−5	0.0339	8.9 × 10−5	0.0495	0.000149
7	Mg2+					1,983.893	1590.948
Rows 8–17 determined with above values for Ca2+ and Mg2+
8	BAPTA	98.8132	119.9513	103.9706	119.9514	89.449	109.663
10	CaBAPTA	21.1868	0.0487	16.0294	0.0486	20.113	0.074
11	MgBAPTA					10.439	10.263
12	EGTA		978.8149		978.8664		586.676
13	CaEGTA		21.1851		21.1336		21.152
14	MgEGTA						392.173
15	Parvalbumin			1.8293	6.9484	0.260	0.379
16	CaParvalbumin			5.1707	0.0516	1.072	0.005
17	MgParvalbumin					5.668	6.617
Differences between values with and without EGTA
18	ΔBAPTA		21.1381		15.9808		20.214
19	ΔCaBAPTA		−21.1381		−15.9808		−20.039
20	ΔMgBAPTA						−0.176
Rows 21–24: Absorbance changes at 292 nm calculated from rows 18–20
21	ΔABAPTA		0.1059		0.0800		0.1013
22	ΔACaBAPTA		−0.0067		−0.0051		−0.0064
23	ΔAMgBAPTA						−0.0005
24	ΔAtotal or A0-AM		0.0992		0.0749		0.0944
Predicted [CaT] = (A0 − AM)/(Δε · l)
25	Predicted CaT		21.151		15.970		20.132
Row 26 gives the value in row 25 divided by that in row 1
26	Predicted/actual		0.996		0.752		0.948

This table provides simulations of experimentally predicted values of [Ca_T] with and without Mg present and with and without metal binding to parvalbumin. The text provides most of the details for the assumed and predicted values. For the case of parvalbumin, Ca and Mg binding was calculated as described with Table 2. Relationships used to calculate Ca and Mg binding to BAPTA and EGTA are given in Section 1 of the supplemental text. For rows 21 and 22, values of 5,009 and 320 M⁻¹ cm⁻¹, respectively, were used for the extinction coefficients at 292 nm of the metal-free and Ca-bound forms of BAPTA (ε_B and ε_CaB, respectively; see Materials and methods). The value of 2,665 M⁻¹ cm⁻¹ assumed for the extinction coefficient for the Mg-bound form of BAPTA (ε_MgB) in row 23 was based on the observation of Tsien (1980) that the decrease in absorbance of BAPTA with Mg binding was about half of that produced by Ca binding (2,665 = 320 + 0.5 × (5,009 − 320)). As used throughout, the value of Δε used to calculate [Ca_T] (row 25) from A₀ − A_M (row 24) was ε_CaB − ε_B, i.e., −4,689 M⁻¹ cm⁻¹ (−4,689 = 320 − 5,009). Row 26 gives the ratios of the values in row 25 to those in row 1.