Overproduction, Purification and Structural Studies
281
5.2
The Presence of Zinc in TthS14 Protein
As has already been mentioned above, the primary structure of this protein leads
to the suggestion that the four cysteine residues are involved in a C4 zinc-finger
motif. The atomic absorption results support this hypothesis, because analysis of
the S14 ribosomal protein from Thermus thermophilus indicated the presence of
Zn ions in the stoicheiometry 1:1 (moles of Zn per mole of TthS14 protein).
Table 20.1 shows the Zn content of TthS14 determined by atomic absorption
under various experimental conditions (in all cases the concentration of the protein was 0.4 mg/ml).
Table 20.1. Atomic absorption measurements for the presence of Zn in ribosomal protein TthS14.
The amount of protein used in all cases was 80 !1g
1. TthS 14: TthS 14 dialysed against deionized H20.
2. TthSI4: TthS14 dialysed against H20, ZnCl2 (lOmglml) and H20 successively.
3. TthSI4: TthS14 dialysed against H20, EDTA (50 mM) and H2 successively.
4. BSA:
Bovine serum albumin dialysed against H2•
5. BSA:
Bovine serum albumin dialysed against H20, ZnCl2 (lOmglml) and H20, successively.
6. TthSI4: TthS14 dialysed against 6 M guanidine-HCl in 20 mM Tris-HCI pH 7.5 and 50 mM
EDTA, 50 mM and H20, successively.
7.:
Zn content of the guanidine-HCI 6 M solution in 20 mM Tris-HCI pH 7.5 and 50 mM
EDTA after dialysis of TthSI4.
8.:
Zn content of the EDTA 50 mM solution in 20 mM Tris-HCl pH 7.5 after dialysis of
TthSI4.
9. TthSI4: TthSI4, denatured as in sample 6, dialysed against 0.57 mM ZnCl2 in Tris-HCI pH 7.5,
0.1 NaCl, twice against 50mM EDTA and H20, successively.
Zn standard solution: 3 ppm -> 0.392 A2l4.5• In all cases where 6 M guanidine-HCl was used a background value of 0.12 ppm Zn was subtracted. In all cases where 50 mM EDTA was used a background
value of 0.08 ppm Zn was subtracted.
Sample
Amount of
Absorbance
Zn
Molar ratio
protein (moles)
(214.5 nm)
ppm
moles
Zn/protein
1. TthS14
11xl0- 9
0.104
0.796
12xlO- 9
2. TthS14
11xlO- 9
22.40
176
2.7xlO- 6
245
3. TthS14
11 X 10- 9
0.095
0.730
12xlO- 9
4. BSA
l.l X 10- 9
0
0
0
0
5. BSA
l.l X 10- 9
37.50
294
4.5xlO- 6
4090
6. TthS14 denatured
11xlO- 9
0.008
0.06
0.9xlO- 9
0.08
7. Guanidine-HCl 6 M
0.061
0.45
6.9xlO- 9
(after dialysis of
denatured TthSI4)
8. EDTA 50 mM (after
0.047
0.35
4.7xlO- 9
dialysis of denatured
TthS14)
9. TthS14 renatured
11 X 10- 9
0.097
0.59
7.4xlO- 9
0.81
281
5.2
The Presence of Zinc in TthS14 Protein
As has already been mentioned above, the primary structure of this protein leads
to the suggestion that the four cysteine residues are involved in a C4 zinc-finger
motif. The atomic absorption results support this hypothesis, because analysis of
the S14 ribosomal protein from Thermus thermophilus indicated the presence of
Zn ions in the stoicheiometry 1:1 (moles of Zn per mole of TthS14 protein).
Table 20.1 shows the Zn content of TthS14 determined by atomic absorption
under various experimental conditions (in all cases the concentration of the protein was 0.4 mg/ml).
Table 20.1. Atomic absorption measurements for the presence of Zn in ribosomal protein TthS14.
The amount of protein used in all cases was 80 !1g
1. TthS 14: TthS 14 dialysed against deionized H20.
2. TthSI4: TthS14 dialysed against H20, ZnCl2 (lOmglml) and H20 successively.
3. TthSI4: TthS14 dialysed against H20, EDTA (50 mM) and H2 successively.
4. BSA:
Bovine serum albumin dialysed against H2•
5. BSA:
Bovine serum albumin dialysed against H20, ZnCl2 (lOmglml) and H20, successively.
6. TthSI4: TthS14 dialysed against 6 M guanidine-HCl in 20 mM Tris-HCI pH 7.5 and 50 mM
EDTA, 50 mM and H20, successively.
7.:
Zn content of the guanidine-HCI 6 M solution in 20 mM Tris-HCI pH 7.5 and 50 mM
EDTA after dialysis of TthSI4.
8.:
Zn content of the EDTA 50 mM solution in 20 mM Tris-HCl pH 7.5 after dialysis of
TthSI4.
9. TthSI4: TthSI4, denatured as in sample 6, dialysed against 0.57 mM ZnCl2 in Tris-HCI pH 7.5,
0.1 NaCl, twice against 50mM EDTA and H20, successively.
Zn standard solution: 3 ppm -> 0.392 A2l4.5• In all cases where 6 M guanidine-HCl was used a background value of 0.12 ppm Zn was subtracted. In all cases where 50 mM EDTA was used a background
value of 0.08 ppm Zn was subtracted.
Sample
Amount of
Absorbance
Zn
Molar ratio
protein (moles)
(214.5 nm)
ppm
moles
Zn/protein
1. TthS14
11xl0- 9
0.104
0.796
12xlO- 9
2. TthS14
11xlO- 9
22.40
176
2.7xlO- 6
245
3. TthS14
11 X 10- 9
0.095
0.730
12xlO- 9
4. BSA
l.l X 10- 9
0
0
0
0
5. BSA
l.l X 10- 9
37.50
294
4.5xlO- 6
4090
6. TthS14 denatured
11xlO- 9
0.008
0.06
0.9xlO- 9
0.08
7. Guanidine-HCl 6 M
0.061
0.45
6.9xlO- 9
(after dialysis of
denatured TthSI4)
8. EDTA 50 mM (after
0.047
0.35
4.7xlO- 9
dialysis of denatured
TthS14)
9. TthS14 renatured
11 X 10- 9
0.097
0.59
7.4xlO- 9
0.81