Radiochemistry

5.8.1 In darkness

The binding of ⁶⁴Cu to the samples incubated in darkness show that Cc can bind Cu²⁺ though not very stable.

Heated Cc-adz binds copper marginally better than Cc alone. Possibly due to formation of the semi-stable N2, N4 side chain chelate. High temperature might result in formation of the stable chelate but also in denaturation of Cc resulting in a stable unspecific binding of copper ions and at 50 °C the later process dominates (figure 4.38).

5.8.2 Low flux

UV irradiation at room temperature and low flux (through the regulating slit in the lamp) for up to an hour did not result in significant binding of copper ions to Cc; only 1 % after two EDTA washes. In stark contrast,

BLG alone had a LE of almost 50 % and adding an adamanzane did not have any effect. A possible explanation is that Cu²⁺ can induce dimerization of BLG through oxidation of a free thiol group [25].

Without UV light, this requires unfolding of the natural protein for instance by heating but it is possible that irradiation could induce the electron transfer without previous unfolding, or simply that UV light can cause the unfolding. In any case, this shows that some vectors are very sensitive to the combination of copper ions and UV light.

Cc-adz binds Cu²⁺ slowly but steady at low flux and room temperature; increasing irradiation time by a factor two, almost doubled LE. The surplus of Cc-adz to ⁽⁶⁴⁾Cu²⁺ was also doubled, but it is difficult to say whether this enhanced chelation since more Cc also blocks more UV light. One of the reasons for a slow reaction could be that N-Cc,N’-CH2COOH[2⁴.3¹]adz has to be decarboxylated before a UV photon can induce chelation. Low concentration is less likely to be the explanation, since almost all the Cu²⁺ can be expected to form Q like semi-stable chelates rapidly with the excess of adamanzane. This is supported by figure 4.39 (explanation follows).

5.8.3 High flux

At 50 °C and high flux, Cc alone has a LE of more than 40 %. A colour shift from red to yellow accompanies this reaction, indicating that not only the apo-protein part of Cc but also the heme has been affected. It is difficult to say what happens, except that somehow an EDTA resistant binding of copper ions is formed, stable enough to keep 30 % of the ⁶⁴Cu bound, even after more than 12 hours of EDTA wash. Obviously, this reaction competes with the adamanzane binding of ⁶⁴Cu, since the copper cannot bind at both places at the same time. As a result, adamanzane might chelate less ⁶⁴Cu than if Cc had not denatured, but likewise less

64Cu is bound to Cc when adamanzanes are present. It is safe to assume that the washout rate of ⁶⁴Cu from Cc is the same whether or not an adamanzane is bound to the Cc because of the large surplus of EDTA during the wash. Therefore, since the washout is larger from pure Cc than from Cc-adz, less ⁶⁴Cu is Cc bound in Cc-adz. Where the Cc-adz samples loose 12-13 %-points of activity from after wash 3 to after wash 7, pure Cc looses 18.9 %-points. The question is then: how much of the ⁶⁴Cu in the Cc-adz samples was Cc bound and how much was Cc-adz bound? To answer this, the washout rates were compared as a function of time (figure 4.39). If Cc is regarded as one compartment of the bound ⁶⁴Cu during the EDTA washes, and the adamanzane as another, then ⁶⁴Cu bound to both Cc and adamanzane should have a curve line. Initially it should be parallel to the Cc washout and then asymptotically change to be parallel to pure adz washout.

However, this is not the case. The washout lines of ⁶⁴Cu from Cc-adz as a function of time are straight lines just as the washout line from Cc alone. Therefore, the ⁶⁴Cu must be bound to one compartment (the

adamanzane) in the Cc-adz samples. This means that the average LE of the adamanzanes is 62 % and 17 % of the activity washes out from the adamanzane from after wash 3 to after wash 7. In comparison, pure Cc has a LE of 48 % and a washout of 40 % from after wash 3 to after wash 7. The pure Cc LE might seem high, but that is because Cc has no competition when binding ⁶⁴Cu. The fact that Cc alone has a LE of 48 %

and still bind none of the ⁶⁴Cu in Cc-adz indicates that the Cu²⁺ rapidly binds to the adamanzane when mixed, probably forming the Q like complex.

A LE of 62 % is still not sufficient after 4 hours irradiation/incubation. Since binding to the adamanzanes does not seem to be the problem but formation of the robust chelate does, it is the chelation process that should be improved. The carboxylic acid arm not used for amide bonding to the vector impedes chelation of Cu²⁺ (figure 5.6). Therefore, either N2 or N4 should be a secondary amine capable of gating the last proton out of the adamanzane cavity. In fact, LE could be limited by the amount of adamanzane where the free arm got removed entirely resulting in a secondary amine. If most of the adamanzane ended with a methyl group as free arm, they might not be able to loose the last proton and fully chelate the copper ion. A secondary amine is especially important when chelation is facilitated by UV light but should also be significant for chelation in darkness; perhaps enough to make adamanzanes better than e.g. DOTA. The extremely low washout rate in large excess of EDTA at pH 7 indicates that the robustness is high enough.

The high LE of pure Cc indicates that the protein has been denatured. The colour change indicates that the Soret band and thus the heme group is involved (figure 4.35). This is in spite of filtering the UV light through a NaBr filter before irradiating the samples. Since the colour change was not observed before removing the slit in the UV lamp, this is probably a result of too high light intensity. If so, the problem could be solved by flash irradiation, which prevents double excitation. The flash irradiation could be repeated several times with seconds apart. Employment of a monochromatic UV lamp would also limit excess photons.

Even with flash irradiation by a monochromatic UV lamp, there is a substantial risk that the vector is denatured [60,179]. However, some proteins are more UV sensitive than others, and especially peptides could be selected for UV resistance making UV light induced chelation feasible for tracer molecule production.