R. Radicella

Labeling of proteins with 125I and experimental determination of its specific activity

The purpose of the present work consists in the standardization of the labeling technique of proteins with 125I and the control of the obtained products, principally their specific activities, in order to utilize them correctly in radioimmunoassays. The quantities of Chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3·6 and 9·6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. In fact, radioiodine not bound to the protein remains in the zone corresponding to the labeled protein, when analyzed by electrophoresis. This complication did not occur when chromatographic analysis was carried out. The knowledge of the real specific activity is important since it is necessary for the determination of the mass of protein which must be put into reaction from the absolute activity of the labeled protein. For these reasons the yields and the corresponding specific activities were determined from ascending chromatograms obtained with 70% methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labeled with the proposed method, the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 ± 4. The non-specific combinations were always smaller than 5%. The possibility of determining the actual specific activity by simple chromatography, shown in the present work, should be considered as one more step towards a methodological standardization of radioimmunoassay procedures.

Kinetics of the phagocytosis of radiogold colloids by the reticuloendothelial system in the rat.

Abstract The kinetics of the phagocytosis of radiogold colloids by the reticuloendothelial system of the rat has been studied theoretically and experimentally, assuming a Michaelis-Menten type kinetics and stating equations which allow the analysis of the phenomenon from a more general point of view. In order to investigate the process with dispersions stabilized by an agent other than gelatine, we determined the disappearance curves from the blood stream of polyvinylpyrrolidone (PVP) protected radiogold colloids with mean particle sizes ranging from 25 to 195 A, by continuous measurement of the radioactivity of the blood, using an extracorporeal blood circulation technique. The results of 114 experiments show that the plot of the half-time of disappearance (T) against the number of injected particles per kg of animal (Nop) gives a straight line. They also show that the maximal rate of phagocytosis is (4.13 ± 0.20) × 1014 particles per kg of animal per minute and that Ks has a value of (2.08 ± 0.16) × 1015 particles per kg of animal. When these results are compared with those obtained with gelatine protected gold colloids, we observe that, in the case of PVP stabilized dispersions, T is generally much shorter for the same value of (Nop). These differences suggest that T is not only influenced by (Nop), but also by the nature of the protective agent. On the other hand, it can be shown that the values of T are not specifically modified by the mean size of the colloidal particles. We discuss these observations and we tentatively explain them as due to the different physicochemical properties of the protective agents which will change ther at of adsorption of the particles on the surface of, and its rate of entrance into, the reticuloendothelial cell.

Physicochemical study of the elctrolytic incorporation of iodine into bovine growth Hormone (BGH)

Abstract The mechanism of iodination of the Bovine Growth Hormone (BGH) by means of the electrolytic method (at pH 7·4) was studied as a function of time and of total iodide to protein ratios. The yield of each labelling process was determined by paper electrophoresis. From these values and the respective iodide to protein molar ratios, the iodination degrees were calculated; frequently, iodination degrees higher than 12 (the maximum theoretically possible), were obtained. When the enzymatic hydrolysis of the samples was carried out, an increase of free iodide was observed, which was more significant at high iodide to protein molar ratios. The quantitative analysis of the chromatograms of the hydrolysates, allowed us to determine the relative amount of monoiodinated tyrosine residues (MIT) and diiodinated tyrosine residues (DIT) for different iodination degrees. It could be shown that for iodination degree of 12, only a 70 per cent of the radioactivity appeared as DIT. The calculation of the relative iodination degree for each molar ratio of iodide to protein, demonstrated that only a 75 per cent of the total available sites at the tyrosine residues admits the incorporation of iodine. Thus, it can be deduced that not all the tyrosine residues are equally accessible for the iodine atom.

The influence of zirconium on the labelling of albumin macroaggregates with 113In

Abstract Physicochemical and biological investigations are described, by which it was demonstrated that the incorporation of 20 μg of Zr(IV) and 4 mg of Na 2 PO 4 H per millilitre of suspension are necessary to fix 113 m In to the albumin macroaggregates (MAA). In this way the separation of the label in vivo , due to the presence of plasma transferrin can be prevented. The preparation of 113 m In labelled MAA was standardized for its utilization as a reproducible tracer for lung scintiscanning.