Table 2. Titration protocol for haem into HusA using four stock solutions.

11 titration points chosen are listed below. Target concentration is diluted by less than 5%, and ligand concentration should span up to at least 5x estimated K_d.

Experiment number	[HusA] μM	Total Cuvette volume (μl)	[Heme] μM	Total vol of haem added	(Haem:HusA)	Haem stock solution (μM)	μl of haem added at each point
1	1.000	500	0.000	0	0	100	0
2	0.996	502	0.398	2	0.4	100	1
3	0.992	504	0.794	4	0.8	100	1
4	0.986	507	1.381	7	1.4	100	2
5	0.978	511	2.153	11	2.2	100	2
6	0.971	515	3.689	15	3.8	500	1
7	0.967	517	5.609	17	5.8	500	1
8	0.965	518	7.529	18	7.8	1000	1
9	0.962	520	11.346	20	11.8	1000	2
10	0.956	523	17.017	23	17.8	1000	3
11	0.954	524	36.069	24	37.8	10000	1