Antonio Caputo

Studies on the structure of hemoglobin I. Physicochemical properties of human globin

A method of preparation of globin from human hemoglobin is reported. The protein so obtained has been submitted to different purity analyses such as electrophoresis, ultracentrifuge, diffusion, and heat-stability behaviour. The data obtained agree in demonstrating the high degree of purity of the protein. The most important physicochemical constants of human globin and its coupling capacity for hematin are reported.

HEMOGLOBIN AND MYOGLOBIN.

Publisher Summary This chapter describes hemoglobin and myoglobin. Hemoglobin and myoglobin are, among all proteins, ones that have been, and are, most actively studied; an enormous number of papers have been published over the past hundred years on all aspects of their properties and behavior. The study of these proteins has gone beyond the interest in their physiological role as oxygen carriers because they represent ideal models for investigating the properties of proteins in general, especially of enzymes. Correspondingly, current knowledge of the structure and function of hemoglobin and myoglobin is far greater than that available for any other protein. In spite of this, however, many questions still remain unsolved regarding the exact molecular mechanisms involved in the function of these proteins. Before discussing the properties of respiratory heme proteins, it is necessary to briefly describe some properties and reactions of their prosthetic group, mainly because the characteristic physiological functions of these proteins arise from the intrinsic reactivity of the heme.

Studies on the structure of hemoglobin: III. Physiochemical properties of reconstituted hemoglobins

Abstract Artificial hemoglobins have been reconstituted from native human globin and the following hemes: mesoheme, deuterheme, chloroheme (or Spriographis heme) and hematoheme. All these differ from protoheme in the side chains of the porphyrin ring at position 2 and 4. Some physical and physiochemical properties of thse hemoglobins have been determined. The spectral properties were similar to those of protohemoglobin. The sedimentation coefficients of all these hemoglobins had values near to 4.0 S. The stability to heat and alkali denaturation decreased in the order: proto-, meso-, deutero-, and hematohemoglobin. The O 2 equilibrium of these unnatural hemoglobins also differed from that of protohemoglobin, being characterized by a decrease of the heme-heme interactions. The results have been correlated with the decrease in affinity of the various modified hemes for globin and suggest that the vinyl groups of the heme play an important role in determining the conformation of the hemoglobin molecule.

Studies on chlorocruorin. I. The oxygen equilibrium of Spirographis chlorocruorin.

Abstract The oxygen equilibrium of pure chlorocruorin obtained from the marine worm Spirographis spallanzanii has been investigated. The effect of pH, temperature, salt concentration, mercurials, and concentration of the pigment on the oxygen affinity and on the shape of the dissociation curves has been studied. The results obtained have been compared with those reported for mammalian hemoglobins under similar conditions.

Studies on the structure of hemoglobin II. Properties of reconstituted protohemoglobin and protoporphyrin-globin

Abstract Human hemoglobin has been reconstituted from protohematin and pure native globin. The reconstituted Hb was the same as the natural pigment in its physico-chemical properties (absorption spectra, isoelectric point, electrophoretic behaviour, sedimentation, diffusion, stability to heat and alkali) and in its oxygen equilibrium (hemeheme interaction, Bohr effect, oxygen affinity). The compound of globin with protoporphyrin was also obtained and investigated. The molecular properties of this protoporphyrin-globin are very similar to those of hemoglobin.

Studies on chlorocruorin: II. Some physicochemical properties of Spirographis chlorocruorin

Abstract Some physicochemical properties of chlorocruorin purified from the blood of Spirographis spallanzanii have been studied. The protein appeared homogeneous both in the ultracentrifuge and in electrophoresis; the sedimentation constant, S 20,w staggered 0 was found to be 57.5 (at neutral pH), and the isoelectric point near 4.2. The molecular weight of Chlorocruorin proved to be about 2.8 × 10 6 from sedimentation-diffusion and from light-scattering measurements. The heme content of the pigment was 1.83%, giving a minimum molecular weight of about 35 × 10 3 . It has also been shown that in concentrated urea solutions and at high pH Chl dissociates into subunits of low molecular weight.

Modulation of adriamycin uptake by lonidamine in ehrlich ascites tumor cells

The effect of Lonidamine, 1-(2,4 dichlorobenzyl)-1-H-indazol-3-carboxylic acid, on the uptake of Adriamycin by Ehrlich ascites tumor cells has been investigated. The uptake of Adriamycin is greatly stimulated by Lonidamine and the increase depends on the energy sources of the cell. In the presence of glucose the intracellular drug content is remarkably lower than that in its absence. This difference lies in the mechanism by which Lonidamine enhances the uptake of Adriamycin. The Adriamycin efflux is via an active transport process and, in the presence of glucose, both aerobic glycolysis and oxidative phosphorylation contribute to ATP synthesis. Although Lonidamine inhibits both these pathways, there is still sufficient ATP to extrude a certain amount of Adriamycin. The elevated intracellular concentration of Adriamycin depends not only on the Lonidamine-inhibited outward transport but also on higher membrane permeability which allows a low concentration of Adriamycin (18 microM) to interfere also with the oxidative metabolism of Ehrlich ascites tumor cells.

The oxygen equilibrium of reconstituted hemoglobins. III. Human mesohemoglobin

Abstract The oxygen equilibrium of mesohemoglobin synthesized from human globin and mesohematin has been studied. It has been found that in mesoHb, the heme-heme interaction is lower than in protoHb and similar to that in deuteroHb. The O 2 affinity of mesoHb is higher than that of proto- and deuteroHb. The effect of pH and temperature on the O 2 equilibrium of mesoHb is analogous to that for protoHb and deuteroHb. These results show that the double bonds in the vinyl groups of the heme play a role in the mechanism of heme-heme interaction in hemoglobin.

The potential role of lonidamine (LND) in the treatment of malignant glioma. Phase II study.

SummaryUp-to-date unsatisfactory results obtained in multimodality treatments of malignant glioma have prompted the research of new therapeutic modalities with ‘unconventional’ modes of action. Lonidamine (LND) is a drug which reduces aerobic glycolytic activity in both human and experimental tumors. This effect mainly depends on the inhibition of mitochondrially-bound hexokinase (HK) which is present in large amounts in malignant cells. A Phase II study was conducted on patients with recurrent glioma; 12 patients were admitted to the study. Clinical side effects were moderate, necessitating a reduction of the dosage in only 1 case. The objective results were evaluated according to the indications of Levin. 2 responders and 3 cases of stable disease were observed out of 10 evaluable patients. The potential value of this new drug is discussed.

Effect of lonidamine on human malignant gliomas: Biochemical studies

SummaryLonidamine (LND) has been shown to inhibit tumor aerobic glycolysis. Its effect was evaluated on several human astrocytomas at different degrees of malignancy; a correlation was found between LDN effect on lactate production and tumor malignancy: in grade I and II astrocytomas LND stimulates lactate production, while in grade III, IV and glioblastoma multiforme lactate production is inhibited.In an attempt to explain this different behaviour, hexokinase content and compartmentation was evaluated in astrocytomas from fresh operatory specimens and from cultured cells as well, observing a significative correlation between malignancy, hexokinase activity, percent of mitochondrially-bound hexokinase and LND effect.The results justify from a biochemical point of view the role of LND as a ‘non-conventional’ agent in multimodality combined treatment for malignant gliomas.