4 Results
4.9 Radiochemistry
4.9.1 N-CH2COOH[24.31]adz-Cc binds 64Cu2+ via the adamanzane when irradiated
To test the UV light induced chelation technique for radiochemical use, adamanzane coupled Cc and pure Cc were irradiated for half an hour with carrier added 64Cu2+ in a closed UV lamp with a light reducing slit (figure 3.2). After irradiation, the samples were ultra-filtrated and washed twice in a large surplus of EDTA (figure 4.36). After the first wash, most of the copper ions had been removed and there was only a minor difference between the Cc and Cc-adz samples, but after the second wash, there was a clear distinction between them. However, the labelling efficiency (LE) was low: approximately 6.5 % (figure 4.36a).
Remaining 64Cu activity after each wash
1%
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Water EDTA 1 EDTA 2
Irradiated Cc-adz 1 Irradiated Cc-adz 2-3 Irradiated Cc-adz 4 Irradiated Cc
Remaining 64Cu activity after each wash
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100%
Water EDTA 1 EDTA 2
Irradiated Cc-adz 1 Irradiated Cc-adz 2 Irradiated Cc Heated Cc-adz 1 Heated Cc-adz 2 Heated Cc
Figure 4.36: Washout of 64Cu labelled Cc and Cc-adz. The numbers of the Cc-adz refer to peaks in the FPLC
separation. a) 10 µM Cc (-adz) was incubated with 1.0 Mbq 1.0 µM (64)Cu(ClO4)2 by irradiation in a 2 mm quartz cuvette for ½ hour by a UV lamp through a slit. The water was removed by ultra-filtration in Centricon tubes. Then the filter was refilled with 1.0 mL 5.0 mM EDTA solution, which was ultra-filtered by centrifugation for 1 hour. The EDTA wash was then repeated. All measurements of 64Cu activity was normalized by comparison to 64Cu standard to
a b
eliminate errors due to decay. b) Same procedure as a) except that [Cc] was 20 µM and irradiation time was 1 hour.
Other samples were not irradiated but incubated at 40 °C for 3 hours instead. Furthermore, most or the entire Cc-adz 2 sample consisted of N-CH2CONH2[24.31]adz-Cc instead of N-CH2COOH[24.31]adz-Cc.
To increase the LE, the irradiation time was doubled to 1 hour in the next experiment and the irradiated samples were compared to samples heated to 40 °C for 3 hours (figure 4.36b). The Cc-adz 2 in this setup was synthesized a bit different so some or all of it was N-CH2CONH2[24.31]adz-Cc instead of
N-CH2COOH[24.31]adz-Cc. As there is no significant difference in LE and washout rate between Cc-adz 1 and Cc-adz 2, this means either that very little of the Cc-adz 2 had an amide end instead of a carboxylic acid end, or that it makes no difference. Another result was that doubling irradiation time almost doubled the LE of Cc-adz to 11 % and that it has negligible effect to heat the samples to 40 °C.
It was then tested, whether increasing the pH would affect the LE, but it did not, and whether ultrasound could induce chelation, which it did not (data not shown).
4.9.2 Irradiated β-Lactoglobulin binds 64Cu2+ independent of the adamanzane
As an alternative to Cc, β-lactoglobulin (BLG) was tested as a model protein for the UV light induced chelation to adamanzanes as BFC. N-CH2COOH[24.31]adz-β-lactoglobulin was synthesized and purified similar to Cc-adz. It was then labelled with 64Cu2+ in the same way as Cc-adz in figure 4.36b. The result was that at 40 °C no chelation or unspecific binding to BLG occurs. Irradiation on the other hand, causes a LE of in average 47 % after the second EDTA wash. Unfortunately, there was no difference between the pure BLG and the BLG-adz samples (figure 4.37). Without irradiation, 64Cu2+ does not bind strong enough to BLG to withstand wash by water. This is in contrast to Cc were water alone is unable to remove 64Cu2+. Irradiation thus increases the copper ion binding ability of BLG from less than that of Cc to higher than that of Cc.
Remaining activity after each wash
1%
10%
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Water EDTA 1 EDTA 2
Irradiated BLG-adz 1 Irradiated BLG-adz 3 Irradiated BLG Heated BLG-adz 1 Heated BLG-adz 3 Heated BLG
Figure 4.37: Washout of 64Cu labelled BLG and BLG-adz. 10 µM BLG (-adz) was incubated with 1.0 Mbq 1.0 µM
(64)Cu(ClO4)2; either by irradiation in a 2 mm quartz cuvette for 1 hour by a UV lamp through a slit, or by heating to 40
°C for 3 hours. The water was removed by filtration in Centricon tubes. Then the tube was refilled with 1.0 mL 5.0 mM EDTA solution that was filtered by centrifugation for 1 hour. The EDTA wash was then repeated. All measurements of
64Cu activity was normalized by comparison to 64Cu standard to eliminate errors due to decay.
4.9.3 Cytochrome c also binds 64Cu2+ if the UV photon flux is too high
In an attempt to increase the LE to more than 50 %, the split in the UV lamp was removed and the samples were irradiated for 2 hours at 50 °C. Furthermore, the washout scheme was changed so the samples were dissolved in 5.0 mM EDTA at pH 7 continuously until the final filtration. Centrifugation for 1 hour removed approximately 90 % of the volume, which then was replaced with new EDTA. Therefore, initially, the unbound 64Cu was diluted, but after two or three washes, the 64Cu in the filtrate was a result of
transchelation, either from the adamanzane or from Cc (figure 4.38).
Increasing the temperature to 50 °C and the incubation time to 4 hours did not cause measurable chelation by the adamanzane without irradiation, as there was no difference between pure Cc and the adamanzane
coupled samples incubated in darkness. Nevertheless, a substantial amount of 64Cu (5-7 %) remained bound to the samples even after 5 EDTA washes. Not even incubation for more than 12 hours with 5 mM EDTA could remove it all, showing that Cc alone can bind Cu2+ with high stability.
Irradiation caused the samples to change colour from red to yellow, indicating a change to the Soret band like the one described in figure 4.35. This happened within the first hour, indicating that it was the increased photon flux due to the split removal rather than the increased duration that caused the colour change. All the irradiated samples had a much higher LE than in earlier experiments. After the third wash where all
uncoordinated Cu2+ can be assumed to be removed, the Cc-adz samples had a LE of 64-71 %, but the pure Cc sample had a LE of 48 %. If it this means that 48 %-points of the 64Cu bound to the Cc-adz samples where bound directly to Cc and not the adamanzane, then the Cc-adz samples should loose 64Cu at a faster rate than pure Cc: The same amount from Cc + a small amount from the adamanzane. However, this is not the case, as Cc alone looses more 64Cu than Cc-adz in the following 3 washes and after the final 12 hour wash, the LE was: Cc-adz: 52-58 %; Cc: 29 %. The difference had increased from 18 to 23 %-points, indicating that less 64Cu was Cc bound in the Cc-adz samples than in the Cc sample.
Remaining 64Cu activity after each wash
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1 2 3 4 5 6 7 (12 h)
Water EDTA
Irradiated Cc-adz 1 Irradiated Cc-adz 3 Irradiated Cc-adz 4 Irradiated Cc Heated Cc-adz 1 Heated Cc-adz 3 Heated Cc-adz 4 Heated Cc
Figure 4.38: Extended washout of 64Cu labelled Cc and Cc-adz. 40 µM Cc-adz/Cc and 2.0 µM (64)Cu(ClO4)2 incubated at 50 °C for 4 hours or irradiated in a 2 mm quartz cuvette by a UV lamp without a slit for 2 hours and incubated for 2 hours at 50 °C. The samples were transferred to Centricon tubes and 2.0 mL water was added. The tubes were centrifuged for 1 hour, filtering the samples. 2.0 mL 4.0 mM EDTA pH 7 was then added to the tubes followed by centrifugation for 1 hour. This was repeated 6 times. The last time, the samples were left over night and were centrifuged for 90 minutes removing all solute. Finally, the protein was dissolved in 2 mL water and transferred to a glass vial to prevent counting of Centricon tube bound activity. All measurements of 64Cu activity was normalized by comparison to 64Cu standard to eliminate errors due to decay.
After the third wash (the second EDTA wash), the EDTA concentration can be assumed constant, and the washout should therefore be a function of time. Interestingly, the amount of bound 64Cu drops in as a logarithmic function (figure 4.39), not as the expected exponential function. This might not be important, as it could be a result of the method, where samples are diluted with fresh EDTA solution instead of complete filtration followed by addition of fresh EDTA solution. Significant is, that all irradiated samples including the Cc control follow logarithmic functions and none of the non-irradiated samples does. This means that the washout from the Cc follows the same pattern as the washout from the adamanzane although much faster.
The Cc function reaches zero after 342 days, where it takes from 59-79 thousand days to reach zero for the Cc-adz samples.
64
Cu washout by EDTA as a function of time
y = -0.1Ln(x) + 0.5835 R2 = 0.9992 y = -0.0701Ln(x) + 0.7906
R2 = 0.9999 y = -0.0657Ln(x) + 0.7218
R2 = 0.9977
y = -0.0642Ln(x) + 0.7083 R2 = 0.9998
0%
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Hours of EDTA treatment
Remaining acivity
Irradiated Cc-adz 1 Irradiated Cc-adz 3 Irradiated Cc-adz 4 Irradiated Cc Heated Cc-adz 1 Heated Cc-adz 3 Heated Cc-adz 4 Heated Cc
Figure 4.39: The EDTA washout from figure 4.37 as a function of time. The first EDTA wash is not included, since the concentration and volume was different from the rest.