Sylvia S.L. Harwig

Macromolecular complexes formed as the result of chloramine-T radioiodination of proteins

Fibrlnogen has recentiy been shown to form macromolecular aggregates during radioiodination with chloramine-T. Decreased half-lives of chloramine-T radioiodinated fibrinogen have also been noted. The effects of chloramine-T on fibrinogen fragment D, fibrinopeptide A, albumin and gamma G globulin were studied to determine if aggregation is unique to fibrinogen. With fragment D and albumin, ICl-labelled protein had identical column behavior to the unlabelled protelns. Chloramine-T labelled protein showed protein products of higher molecular weight than native Fg-D. Albumin was reacted with varylng amounts of chloramine-T in the absence of iodide. In high C-T concentrations, there was significant production of higher molecular weight species. These results suggest that use of standard chloramine- T radioiodination may result in macromolecular species which would be unsuitable for in vlvo purposes. It would seem desirable that any protein labelled with chloramine-T for in vivo studies should be examined for the presence of this type of subtle denaturation prior to use. (auth)

Effect of iodination level on the properties of radioiodinated fibrinogen

Abstract The effect of iodination level on the biological clearance of radioiodinated fibrinogen was investigated. Canine, human and rabbit fibrinogen were included in the study to also test for possible differences among various species. Iodination of fibrinogen with 3 to 9 iodine atoms/molecule resulted in essentially unchanged isotopic clottability as compared to preparations at the conventional level of 0.5 iodine atom/molecule. No changes in molecular size due to covalent aggregation were observed by SDS gel electrophoresis with radioiodinated human fibrinogen preparations. Canine and human fibrinogen iodinated with up to 4.5 iodine atoms/molecule exhibited little change in biological clearance behavior relative to the conventional iodination level. However, rabbit fibrinogen iodinated with 3.5 iodine atoms/molecule displayed a difference in clearance when compared to the 0.5 iodine atom/molecule preparations. Thus, canine and human fibrinogen can be iodinated with 4.5 iodine atoms/molecule without altering their biological clearance behavior while rabbit fibrinogen is more sensitive to this iodination level.

Preparation and in vitro properties of 99mTc-fibrinogen

Abstract We have developed a simple, mild and efficient electrolytic labeling technique for preparing 99mTc-fibrinogen for use in the radioisotopic detection of deep vein thrombosis. The method employs tin electrodes with in situ generation of Sn2+ as intermediate reducing agent for 99mTcO4−. Wide differences in labeling behavior are observed as the reaction conditions are varied. The optimum labeling system consists of 2 mg fibrinogen and the desired level of Na 99mTcO4 in 2 ml 0.01 M phosphate-0.15 M NaCl buffer, pH 6.0. The 99mTc-fibrinogen labeling efficiency is 70–80%, with a total preparation time of ca. 25 min. Sepharose-4B gel chromatography indicates that the non-protein bound radioactivity is in the form of a chelate derived from anions of the buffer system, with no free unreduced 99mTcO4− present. The labeled fibrinogen is easily separated and purified by ammonium sulfate precipitation and shows good in vitro stability. The isotopic clottability of the product is 50–65%, depending on the species of fibrinogen. When prepared by the method reported here, 99mTc-fibrinogen appears to be a potentially useful radiopharmaceutical for external scintillation imaging of deep vein thrombi in patients.

123I-labeled soluble fibrin: preparation and comparison with other thrombus imaging agents.

Abstract Radioiodinated soluble fibrin, a derivative of radioiodinated fibrinogen, has been investigated for thrombus localization. Soluble fibrin can be labeled with 123 I as the radionuclide for imaging thrombi with a scintillation camera. Protein iodination grade 123 I is produced by a distillation and extraction procedure from the product of the 121 Sb ( α , 2 n ) 123 I reaction in the Washington University 54-in. cyclotron. Labeling with these 123 I preparation is effected by the iodine monochloride method. Induced 4–6-hr-old femoral vein thrombi in dogs can be visualized beginning 2 hr after injection of 1 mCi of 123 I-labeled soluble fibrin. This new radiopharmaceutical may be useful for imaging actively-propagating thrombi in patients, especially in areas of large blood pool. Its potential appears to be similar to that of 123 I-labeled highly iodinated fibrinogen, another new thrombus imaging agent.

Solid phase bovine thrombin. Preparation and properties.

A new solid-phase thrombin (EC 3.4.21.5) was prepared through conjugation of the enzyme under mild conditions to a glass support bearing an active ester of N-hydroxysuccinimide. The immobilized enzyme retained 50 +/- 10% of the specific esterase activity of the parent soluble enzyme. The Km (apparent) for the esterase activity of the immobilized enzyme has a value of 5 mM, identical of the Km value of the parent-soluble enzyme. Only 6 +/- 1% of the specific proteolytic activity was retained and a higher Km (apparent) value of 67 muM was obtained for the insoluble enzyme compared to Km value of 12.5 muM for the parent soluble thrombin. Solid-phase thrombin prepared by the diazocoupling technique was previously reported to retain only 3% of the specific proteolytic activity. The observed loss of specific proteolytic activity can be attributed to steric interference, a change in charge characteristics, or both. Nevertheless, the present method of preparation has the advantages of rapidity and simplicity. It can readily be adapted to use for studying the fate of various complexes of fibrinogen, fibrin and their degradation products. It should also be useful for preparing radiolabeled autologous soluble fibrin for thrombus detection in patients undergoing active thrombosis.