282
P. TSIBOLI et al.
5.2.1
Atomic Absorption of the Overproduced Protein
As we mentioned above, atomic absorption experiments indicated the presence
of Zn ions in the stoicheiometry 1:1 (moles of Zn per mole of TthS14 protein). In
order to study the behaviour of the protein against Zn, the protein was dialysed
against ZnClz in a concentration of 10 mg/ml for 3-4 hrs, followed by another
dialysis against water (with two changes,the first lasting 2 hrs and the second 16
hrs) in order to remove traces of ZnCh About 245 moles of Zn per mole of S14
were absorbed onto the surface of the protein (Table 20.1, sample 2). A similar
behaviour is shown by albumin (Table 20.1, sample 5), which was subjected to the
same treatment.
5.2.2
Attempts for Zn Detachment Without Denaturation
To study the "affinity" of Zn to the protein we used EDTA - as chelating agent -
or acidic conditions as described below.
In an attempt to remove the Zn bound to TthS14 the protein was dialysed
extensively against water (16 h), against 20 mM Tris-HCl pH 7.5 buffer containing
50 mM EDTA - as chelating agent - (8 h) and once more against water (16 h),
successively. In this case the measured zinc content is the same as that of the protein dialysed only against water, i.e. a Zn-protein molar ratio of one was calculated, indicating that EDTA alone is not enough to remove the Zn ion bound to
TthS14 (Table 20.1, Sample 3).
It was also attempted to remove Zn under acidic conditions because exposure
to acid pH has been shown to remove Zn from zinc-finger proteins (Woods et ai.
1995). However, dialysis of TthS14 against pH 5.0 failed to remove the Zn ion
from the protein. The Zn-protein stoicheiometry remained 1:1. This indicates a
stronger than usual affinity of the TthS14 protein for the zinc ion. Fifty mM
EDTA could not be used at this pH (5.0) because it is barely soluble at room temperature at 25°C and precipitation might occur at 4°C-where all the Zn detachment procedures took place.
5.2.3
Zn Detachment Under Denaturating Conditions
Now the question arises "How does Zn bind to the protein? Is it covalently bound
or absorbed onto the surface of the protein?".
In an attempt to remove the Zn bound to TthS14, the protein was denatured by
dialysis against 6 M guanidine hydrochloride in 20 mM Tris-HCl pH 7.5, 50 mM
EDTA (the buffer volume was 500 ml and the duration of the dialysis 4 h), followed by dialysis against 500 ml 50 mM EDTA in 20 mM Tris-HCl pH 7.5 for 4 h
and then against water (16 h). In this case the drastic disturbance of the protein
conformation with 6 M guanidine hydrochloride, followed by dialysis against 50
mM EDTA and finally against water, leads to an almost complete removal of Zn
(0.06 ppm) as shown in Table 20.1, Sample 6. On the other hand, the guanidine
P. TSIBOLI et al.
5.2.1
Atomic Absorption of the Overproduced Protein
As we mentioned above, atomic absorption experiments indicated the presence
of Zn ions in the stoicheiometry 1:1 (moles of Zn per mole of TthS14 protein). In
order to study the behaviour of the protein against Zn, the protein was dialysed
against ZnClz in a concentration of 10 mg/ml for 3-4 hrs, followed by another
dialysis against water (with two changes,the first lasting 2 hrs and the second 16
hrs) in order to remove traces of ZnCh About 245 moles of Zn per mole of S14
were absorbed onto the surface of the protein (Table 20.1, sample 2). A similar
behaviour is shown by albumin (Table 20.1, sample 5), which was subjected to the
same treatment.
5.2.2
Attempts for Zn Detachment Without Denaturation
To study the "affinity" of Zn to the protein we used EDTA - as chelating agent -
or acidic conditions as described below.
In an attempt to remove the Zn bound to TthS14 the protein was dialysed
extensively against water (16 h), against 20 mM Tris-HCl pH 7.5 buffer containing
50 mM EDTA - as chelating agent - (8 h) and once more against water (16 h),
successively. In this case the measured zinc content is the same as that of the protein dialysed only against water, i.e. a Zn-protein molar ratio of one was calculated, indicating that EDTA alone is not enough to remove the Zn ion bound to
TthS14 (Table 20.1, Sample 3).
It was also attempted to remove Zn under acidic conditions because exposure
to acid pH has been shown to remove Zn from zinc-finger proteins (Woods et ai.
1995). However, dialysis of TthS14 against pH 5.0 failed to remove the Zn ion
from the protein. The Zn-protein stoicheiometry remained 1:1. This indicates a
stronger than usual affinity of the TthS14 protein for the zinc ion. Fifty mM
EDTA could not be used at this pH (5.0) because it is barely soluble at room temperature at 25°C and precipitation might occur at 4°C-where all the Zn detachment procedures took place.
5.2.3
Zn Detachment Under Denaturating Conditions
Now the question arises "How does Zn bind to the protein? Is it covalently bound
or absorbed onto the surface of the protein?".
In an attempt to remove the Zn bound to TthS14, the protein was denatured by
dialysis against 6 M guanidine hydrochloride in 20 mM Tris-HCl pH 7.5, 50 mM
EDTA (the buffer volume was 500 ml and the duration of the dialysis 4 h), followed by dialysis against 500 ml 50 mM EDTA in 20 mM Tris-HCl pH 7.5 for 4 h
and then against water (16 h). In this case the drastic disturbance of the protein
conformation with 6 M guanidine hydrochloride, followed by dialysis against 50
mM EDTA and finally against water, leads to an almost complete removal of Zn
(0.06 ppm) as shown in Table 20.1, Sample 6. On the other hand, the guanidine