TABLE 2.

Metals present in the recombinant CBD_CelK and its mutated variants

Variant	Amt of metal/mole of proteina
	Ca	Cu	Zn
CBDCelKb	1.03	0.101	0.095
CBDCelK (CaCl2)c	1.11	0.091	0.083
CBDCelK (EDTA)d	0.83	0.065	0.073
CBDCelK (urea-EDTA)e	0.64	0.072	0.042
W56Ab	0.87	0.051	0.063
W94Ab	1.02	0.087	0.056
Y111Ab	0.91	0.066	0.087
Y136Ab	1.11	0.076	0.088
D192Ab	0.07	NDf	ND
D192A (CaCl2)c	0.07	ND	ND

^aMetal content was determined by plasma emission spectroscopy. The results shown are averages of three determinations. In all variants, 20 mM Tris-HCl buffer (pH 7.5) was used. After incubation of proteins with CaCl₂ or EDTA at 4°C overnight, they were extensively dialyzed against Tris buffer without additives. 

^bProtein was dialyzed three times against 1 liter of Tris buffer. 

^cProtein was incubated with 5 mM CaCl₂ and then dialyzed. 

^dProtein was incubated with 5 mM EDTA and then dialyzed. 

^eProtein was incubated with 5 mM EDTA in buffer containing 8 M urea and then dialyzed. 

^fND, not detected.