Separation of 44Ti from proton irradiated scandium by using solid-phase extraction chromatography and design of 44Ti/44Sc generator system.

Scandium-44g (half-life 3.97h [1]) shows promise for positron emission tomography (PET) imaging of longer biological processes than that of the current gold standard, 18F, due to its favorable decay parameters. One source of 44gSc is the long-lived parent nuclide 44Ti (half-life 60.0 a). A 44Ti/44gSc generator would have the ability to provide radionuclidically pure 44gSc on a daily basis. The production of 44Ti via the 45Sc(p,2n) reaction requires high proton beam currents and long irradiation times. Recovery and purification of no-carrier added (nca) 44Ti from scandium metal targets involves complex separation chemistry. In this study, separation systems based on solid phase extraction chromatography were investigated, including branched diglycolamide (BDGA) resin and hydroxamate based ZR resin. Results indicate that ZR resin in HCl media represents an effective 44Ti/44gSc separation system.

Large scale accelerator production of 225Ac: Effective cross sections for 78-192MeV protons incident on 232Th targets.

Actinium-225 and 213Bi have been used successfully in targeted alpha therapy (TAT) in preclinical and clinical research. This paper is a continuation of research activities aiming to expand the availability of 225Ac. The high-energy proton spallation reaction on natural thorium metal targets has been utilized to produce millicurie quantities of 225Ac. The results of sixteen irradiation experiments of thorium metal at beam energies between 78 and 192MeV are summarized in this work. Irradiations have been conducted at Brookhaven National Laboratory (BNL) and Los Alamos National Laboratory (LANL), while target dissolution and processing was carried out at Oak Ridge National Laboratory (ORNL). Excitation functions for actinium and thorium isotopes, as well as for some of the fission products, are presented. The cross sections for production of 225Ac range from 3.6 to 16.7mb in the incident proton energy range of 78-192MeV. Based on these data, production of curie quantities of 225Ac is possible by irradiating a 5.0gcm-2 232Th target for 10 days in either BNL or LANL proton irradiation facilities.

The effect of calcium ion on the urea denaturation of immobilized bovine trypsin.

The effect of calcium ion on the urea denaturation of trypsin has been investigated. By using trypsin immobilized on glass beads, all possibilities of autolysis occurring during the denaturation process are eliminated. It was found that in 8 M urea calcium ion markedly decreases the denaturation rate of the immobilized trypsin. Conversely, the presence of calcium ion markedly accelerates the rate of renaturation of denatured immobilized trypsin. Calcium may exert its stabilizing effect on the tertiary structure of the protein by coordination to the side chains of Asp 194, Ser 190 and the carbonyl group of Ser 139 (using the chymotryptic numbering system).

Tyrosine fluorescence as a measure of denaturation in thermolysin.

The heat and guanidine hydrochloride denaturation of thermolysin has been followed by fluorescence techniques. The native enzyme has a single emission peak which is decreased in intensity and which splits into two clearly resolved peaks upon denaturation. These data are interpreted to indicate that energy transfer from tyrosine to tryptophan occurs in the native enzyme which is lost upon denaturation. Even though zinc is fully bound to thermolysin at 90 degrees C or in the presence of 6 M guanidine hydrochloride, removal of zinc from the denatured enzyme has no effect on the emission spectrum. Removal of Ca2+ from the denatured enzyme. These data indicate that even though the metal ions are bound to the denatured protein, they provide little structural integrity to the protein as measured by energy transfer between tyrosine and tryptophan.

Lanthanide-binding properties of rat oncomodulin.

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.

Application of ion exchange and extraction chromatography to the separation of actinium from proton-irradiated thorium metal for analytical purposes.

Actinium-225 (t1/2=9.92d) is an α-emitting radionuclide with nuclear properties well-suited for use in targeted alpha therapy (TAT), a powerful treatment method for malignant tumors. Actinium-225 can also be utilized as a generator for (213)Bi (t1/2 45.6 min), which is another valuable candidate for TAT. Actinium-225 can be produced via proton irradiation of thorium metal; however, long-lived (227)Ac (t1/2=21.8a, 99% ß(-), 1% α) is co-produced during this process and will impact the quality of the final product. Thus, accurate assays are needed to determine the (225)Ac/(227)Ac ratio, which is dependent on beam energy, irradiation time and target design. Accurate actinium assays, in turn, require efficient separation of actinium isotopes from both the Th matrix and highly radioactive activation by-products, especially radiolanthanides formed from proton-induced fission. In this study, we introduce a novel, selective chromatographic technique for the recovery and purification of actinium isotopes from irradiated Th matrices. A two-step sequence of cation exchange and extraction chromatography was implemented. Radiolanthanides were quantitatively removed from Ac, and no non-Ac radionuclidic impurities were detected in the final Ac fraction. An (225)Ac spike added prior to separation was recovered at ≥ 98%, and Ac decontamination from Th was found to be ≥ 10(6). The purified actinium fraction allowed for highly accurate (227)Ac determination at analytical scales, i.e., at (227)Ac activities of 1-100 kBq (27 nCi to 2.7 µCi).

Radioarsenic from a portable (72)Se/(72)As generator: a current perspective.

Positron emission tomography (PET) of slower biological processes calls for the use of longer lived positron emitting radioisotopes. Beyond radionuclide production considerations, practicality and rapidity of subsequent labeling chemistry further limits the selection of radioisotopes with potentially favorable nuclear properties. One additional limitation is the availability of PET radiotracers at the point-of-care with appropriate on-site production methodologies or robust radionuclide generator systems. The positron emitter (72)As (half-life 26 h) is generated via decay of (72)Se (half-life 8.5 d); this pair comprises and excellent generator system for clinical availability of a longer lived PET isotope. Many (72)Se/As generator systems have been introduced utilizing the rich interplay of Se(IV)/Se(VI) and As(III) /As(V) chemical behavior. This paper describes available generator concepts, and briefly outlines some current arsenic labeling methodologies for the introduction of radioarsenic into biomolecules.

Selenium-72 formation via nat Br(p,x) induced by 100 MeV protons: steps towards a novel 72Se/72As generator system.

Selenium-72 production by the proton bombardment of a natural NaBr target has been successfully demonstrated at the Los Alamos National Laboratory Isotope Production Facility (LANL-IPF). Arsenic-72 (half life 26 h) is a medium-lived positron emitting radionuclide with the major advantage of being formed as the daughter of another "generator" radioisotope (Se-72, 8.5 d). A (72)Se/(72)As generator would be the preferred mechanism for clinical utilization of (72)As for positron emission tomography (PET). No portable (72)Se/(72)As generator system has been demonstrated for convenient, repeated (72)As elution ("milking"). In this work, we describe (72)Se production and recovery from irradiated NaBr targets using a 100 MeV proton beam. We also introduce an (72)As generator principle based on (72)Se chelation followed by liquid-liquid extraction, which will be transferred to a solid-phase sorption/elution system.

Proton-induced cross sections relevant to production of 225Ac and 223Ra in natural thorium targets below 200 MeV.

Cross sections for (223,)(225)Ra, (225)Ac and (227)Th production by the proton bombardment of natural thorium targets were measured at proton energies below 200 MeV. Our measurements are in good agreement with previously published data and offer a complete excitation function for (223,)(225)Ra in the energy range above 90 MeV. Comparison of theoretical predictions with the experimental data shows reasonable-to-good agreement. Results indicate that accelerator-based production of (225)Ac and (223)Ra below 200 MeV is a viable production method.

225Ac and 223Ra production via 800 MeV proton irradiation of natural thorium targets.

Cross sections for the formation of (225,227)Ac, (223,225)Ra, and (227)Th via the proton bombardment of natural thorium targets were measured at a nominal proton energy of 800 MeV. No earlier experimental cross section data for the production of (223,225)Ra, (227)Ac and (227)Th by this method were found in the literature. A comparison of theoretical predictions with the experimental data shows agreement within a factor of two. Results indicate that accelerator-based production of (225)Ac and (223)Ra is a viable production method.

Oncomodulin and parvalbumin. A comparison of their interactions with europium ion.

The 7F0----5D0 transition of Eu3+ was used to probe the metal-binding domains of rat oncomodulin and rat parvalbumin. Two distinct differences between the two proteins were observed. The first relates to the pH-dependent behavior of their 7F0----5D0 spectra, a phenomenon noted previously for other paravalbumins. In the case of rat parvalbumin, the spectral features associated with both metal-binding sites titrate concomitantly (pK alpha = 8.2); however, in the case of oncomodulin, the two sites titrate sequentially (pK alpha = 6.3 for the CD site; pK alpha = 8.3 for EF site). The proteins also contrast with regard to their discrimination for Eu3+ over Ca2+. The CD and EF sites in rat parvalbumin both display a large preference for Eu3+: (KCa/KEu)CD = 143 +/- 11 and (KCa/KEu)EF = 191 +/- 30. However, in the case of oncomodulin, although the EF site of oncomodulin greatly prefers the trivalent lanthanide ion (KCa/KEu = 300 +/- 80), the CD site exhibits a relatively minor preference (KCa/KEu = 11 +/- 1).

Resonance energy transfer between cysteine-34, tryptophan-214, and tyrosine-411 of human serum albumin.

Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.

Anthraniloyl-tyrosine 411 as a spectroscopic probe of fatty acid binding to human serum albumin.

Reaction of human serum albumin with p-nitrophenylanthranilate results in transesterification of the anthraniloyl group to tyrosine 411. Titration of anthraniloyl-Tyr-411-albumin with long chain or short chain fatty acids produces marked changes in the absorption and fluorescence spectra of the anthraniloyl moiety as fatty acids bind in the channel near it. It appears that the anthraniloyl group is a very sensitive probe that can follow binding of small molecules at the 3-AB subdomain of human serum albumin.

Synthesis of a new chelating gel: removal of Ca2+ ions from parvalbumin.

The synthesis of a chelating gel which contains the effective metal chelating agent ethylenediaminetetraacetic acid covalently linked to amino-agarose is described. This gel is shown to be a rapid and extremely effective material for the removal of tightly bound, but labile metal ions from proteins without introducing contaminants into the biological system. The synthesis involves the formation of an amide linkage between the dangling carboxylate arm of the [Co(EDTA)Cl]2-complex and amino-agarose using a standard carbodiimide coupling reaction. The chelating gel is shown to remove approximately 98.5% of the calcium from fully bound Ca2-parvalbumin and over 99% of the europium from Eu2-parvalbumin.

Synthesis of affinity-label chelates: a novel synthetic method of coupling ethylenediaminetetraacetic acid to amine functional groups.

An affinity-label chelate for the enzyme trypsin was synthesized by a novel synthetic technique which takes advantage of the presence of a dangling carboxylate arm in the [Co(EDTA)Cl]2- complex anion. The dangling carboxylate group was coupled to the amino group of p-aminobenzamidine, an effective inhibitor of trypsin activity, via the carbodiimmide reaction to produce a trypsin affinity label at one end and a strong EDTA-like chelating agent at the other, coupled through an amide bond. The cobalt ion can be removed if desired by reduction with Fe2+ + ascorbate, and alternate metal ions inserted in its place. The reaction is general, and affinity labels which contain amino groups can be easily coupled via this procedure, allowing the introduction of a paramagnetic or fluorescent probe into a protein or nucleotide system. The same method has been used to prepare a highly effective chelating gel which is capable of removing calcium and lanthanide ions from the binding protein parvalbumin.

Luminescence studies of lanthanide ion binding to parvalbumin.

Luminescence methods were used to examine the interaction of Eu(III) and Tb(III) with parvalbumin isozyme III from pike (Esox lucius). The bound lanthanide ions were excited both directly, via laser irradiation, and indirectly, via fluorescence energy transfer from adjacent phenylalanine residues. At high (175 microM) protein concentrations, the lanthanide titration curves exhibited pronounced quenching of luminescence at Ln3+:parvalbumin ratios above 2:1, in agreement with earlier reports (Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13, 4575-4579). However, in experiments performed with lower concentrations (10 microM), the titrations were well behaved and indicated a lanthanide:protein stoichiometry of 2:1. Equilibrium dialysis measurements performed with Eu(III) ruled out the existence of a third strong binding site which could cause the quenching of the luminescence at high protein concentrations. Similarly, careful analysis of the spectrum that results from direct excitation of the 7F0----5D0 transition of parvalbumin-bound Eu3+ ion revealed no peak attributable to a third Ln3+-binding site. The peak which has been construed by others (Rhee, M.-J., Sudnick, D. R., Arkle, V. K., and Horrocks, W. DeW., Jr. (1981) Biochemistry 20, 3328-3334) as evidence for a third site was shown to result from a pH-dependent spectral transition involving the europium ions bound at the CD and EF sites. Luminescent lifetime measurements performed on Tb(III)/parvalbumin solutions follow Stern-Volmer quenching kinetics at terbium:protein ratios in excess of 2:1, suggesting that the quenching results from collisional deactivation of the tightly bound ions by excess terbium ion free in solution.

The location of the calcium ion binding site in bovine alpha-trypsin and beta-trypsin using lanthanide ion probes.

The effect of Gd3+ on the nuclear magnetic resonance (NMR) relaxation rates, T1m-1 and T2m-1, of inhibitor protons in metal-inhibitor-trypsin ternary complexes has been measured. The Solomon-Bloembergen equations have been used to calculate distances of 10.0 +/- 0.5, 8.8 +/- 0.5, and 9.5 +/- 0.5 A between the metal ion and the methyl and ortho protons of p-toluamidine, and the methyl protons of acetamidine, respectively. Essentially the same results are obtained for both alpha-trypsin and beta-trypsin. Binding constants of 3.3 x 10(3) and 4.1 x 10(3) M-1 for the association of Gd(III) with alpha-trypsin and beta-trypsin, respectively, in the presence of p-toluamidine at pH 6.0 have been obtained by equilibrium dialysis. Calcium binding constants of 260 and 3700 M-1 at pH 6.0 and 8.0, respectively, with beta-trypsin have also been obtained. Calcium ion and gadolinium ion compete for the same site on the protein. Calcium has been shown to protect alpha-trypsin from further autolytic degradation to psi-trypsin. From examination of the crystal structure of the enzyme we propose that the calcium ion binding site of bovine trypsin is comprised of the side chains of Asp-194 and Ser-190 (based on the chymotrypsin sequence numbering system). This seems to be the only site which is comprised of at least one carboxyl group; which fits our distance requirements and which is conisistent with other chemical data.

Eu3+ luminescence studies of oncomodulin. The origin of the pH-dependent behavior.

The Eu3+ 7F0----5D0 excitation spectra of parvalbumin and oncomodulin are pH-dependent. Until now, it had been assumed that both the CD and EF ion-binding sites shared this property and that deprotonation of water molecules coordinated to the bound Eu3+ ions might be responsible for the pH dependence. Results obtained with the site-specific variant of oncomodulin known as D59E, in which glutamate replaces the aspartate naturally present at position 59, have necessitated substantial revision of these ideas. It now appears that the pH-dependent behavior is confined to the CD site. Moreover, we observe no corresponding change in the number of O-H oscillators coordinated to the bound Eu3+ ions in the pH range over which we observe the spectroscopic alteration. It is likely that the behavior results from deprotonation of one or more carboxyl groups clustered at the COOH-terminal end of the CD domain.

Fluorescence energy-transfer measurements between the calcium binding site and the specificity pocket of bovine trypsin using lanthanide probes.

Using fluorescence energy-transfer experiments we have measured the distance between the specificity pocket and the calcium ion binding site of bovine pancreatic trypsin. Proflavin and thionine were used to block the specificity site, whereas various lanthanide ions were substituted for the calcium. It was then possible to choose various donor-acceptor pairs which exhibit suitable energy transfer. We have calculated the distance between proflavin and Nd(III), Pr(III), and Ho(III) to be 10.9, and 10.3, and 10.3 A, respectively. This agrees very well with the value of approximately 10 A we obtained between the methyl protons of p-toluamidine (a competitive inhibitor) and Gd(III) using nuclear magnetic resonance techniques (Abbott, F., Gomez, J.E., Birnbaum, E.R., and Darnall, D.W. (1975), Biochemistry 14, 4935). This is strong evidence that, in solution, the calcium binding site is composed of the side chains of Ser-190 and Asp-194.