The Zn- or Cu-Thionein Character of a Metallothionein Determines Its Metal Load When Synthesized in Physiological (Metal-Unsupplemented) Conditions

Table 2

Metal-to-protein ratios and molecular masses of the recombinant Zn(II)-MT, complexes obtained from the pGEX expression system with no metal supplementation to the culture, except in the Zn-MtnB used as a control (see Section 2). All the measurements have been performed at least twice, from independently obtained samples.

MT protein	Zn/MT(a)	Zn/MT(a)	Zn-MT species(b)	𝑚^(exp^c)	𝑚th^(d)
	(ICP-AES)	(acidic ICP-AES)	(ESI-MS)
mMT1	6.95	7.40	Zn7-mMT1	6605.5	6605.96
Crs5	4.20	4.61	Zn4-Crs5	7715.5	7718.92
			Zn3-Crs5 ⋅ Zn5-Crs5	7654.5 ⋅ 7780.5	7655.53 ⋅ 7782.31
			Zn2-Crs5 ⋅ Zn6-Crs5	7590.4 ⋅ 7844.5	7592.13 ⋅ 7845.70
			Zn1-Crs5 ⋅ Zn7-Crs5	7525.0 ⋅ 7907.5	7528.75 ⋅ 7909.09
MtnB	2.04	2.40	apo-MtnB	4667.2	4669.34
			Zn1-MtnB	4733.8	4732.73
			Zn3-MtnB	4857.0	4859.51
			Zn2-MtnB	4795.0	4796.12
			Zn4-MtnB	4920.8	4922.9
MtnA	1.70	1.94	apo-MtnA ⋅ Zn1-MtnA	3996.4 ⋅ 4059.4	3997.49 ⋅ 4060.88
			Zn2-MtnA	4121.2	4124.27
			Zn3-MtnA	4186.4	4187.66
Zn-MtnB(e)	3.95	5.10	Zn4-MtnB	4922.0	4922.90
			Zn3-MtnB	4850.2	4859.51

ICP-AES inductively coupled plasma atomic emission spectroscopy, ESI-MS electrospray ionization mass spectrometry.
(a)In all experiments Cd and Cu contents were also measured but their amount was always under detection limits.
(b)Species proposed according to the mass difference between holo-protein and apo-protein. Species in bold are the major components of the preparation.
(c)Experimental molecular masses. Measurements were always performed in duplicate. All corresponding standard deviations were always less than 1%.
(d)Theoretical molecular mass of the corresponding species.
(e)Control experiment: MtnB synthesized in Zn(II)-supplemented medium.