Alessandro Rossi-Fanelli

The properties and interactions of the isolated α and β chains of human haemoglobin: I. Sedimentation and electrophoretic behaviour

The α and β chains of human haemoglobin A 1 may be isolated by starch block electrophoresis or by chromatography after treatment of the protein with p -mercuribenzoate. The chains so isolated do not show any free SH groups; however, after treatment with cysteine or thioglycolate, between 1 and 2 SH groups per β chain and 1 per α chain become titratable. The chains before and after such treatment are designated α PMB† or β PMB and α SH or β SH respectively. Ultracentrifugal analysis shows that the isolated α SH and α PMB chains are both monomeric; in contrast the β PMB chains tend to polymerize. The β SH chains are heterogeneous but the major component appears to be a tetramer and may be identified with haemoglobin H. A mixture of α PMB and β PMB chains in equal amounts is homogeneous and has a sedimentation coefficient in the neighbourhood of 3. A similar mixture of α SH and β SH chains behaves like haemoglobin A both in the ultracentrifuge and in starch gel electrophoresis. Certain systems which are homogeneous in the centrifuge and on Sephadex show multiple bands in electrophoresis, which implies differences beyond those due to molecular weight. These results are discussed in terms of the interactions between the chains under various conditions.

The interaction of bromthymol blue with hemoglobin and its effect on the oxygen equilibrium

Abstract The equilibria and kinetics of the reaction of the dye bromthymol blue with various derivatives of human hemoglobin, as well as with several other proteins, have been studied. In the case of normal hemoglobin the affinity of the dye is much greater for the deoxy than for the oxy form and this difference is reflected in the kinetics of the reaction. Conversely the dye has a marked effect on the oxygen equilibrium. The characteristic difference of behaviour shown by the oxy and deoxy forms of normal hemoglobin is lacking in myoglobin and those modified forms of hemoglobin which have simple hyperbolic oxygen-equilibrium curves.

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.

The properties and interactions of the isolated alpha- and beta-chains of human haemoglobin. V. The reaction of alpha- and beta-chains.

The mixing of the isolated α - and β -chains of human haemoglobin is accompanied by spectroscopic changes in the Soret region and by changes in the kinetics of the reaction with ligands and in sedimentation behaviour. These changes show that when the chains are combined with p -mercuribenzoate, the system is in a state of labile association—dissociation equilibrium which is strongly dependent on pH between pH 7 and 8, and is oxygen linked. The kinetics of the spectroscopic changes reveal that, both in the presence and absence of p -mercuribenzoate in the chains, the recombination is a complex process with an over-all half-time of the order of one to five seconds at 30°C, when the chains are at micromolar concentration.

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.

Enzymic reduction of ferrimyoglobin

Abstract The following enzymic systems capable of reducing Mb+ have been studied: 1. 1. DPN reductase isolated from the heart of pig, which reduces Mb+ only in the presence of methylene blue. 2. 2. TPN reductase prepared from yeast. 3. 3. Cytochrome b2 which slowly reduces Mb+ both in anaerobic and aerobic conditions. The addition of quinone augments enormously the velocity of the reduction of Mb+ by cytochrome b2. 4. 4. Even the DPNH-quinone and TPNH-quinone systems are able to reduce Mb+ in various experimental conditions. A comparative study of reduction velocities by various enzymic systems of cytochrome c, Mb+, and Hb+ demonstrates that these three pigments are reduced at different rates. The difference in the velocity of the reduction of Mb+ and Hb+ represents another characteristic distinction between these two pigments.

Studies on chlorocruorin: III. Electron microscope observations on Spirographis chlorocruorin

The structure of chlorocruorin from the marine worm Spirographis spallanzanii has been investigated by electron microscopy. The molecule (mol. wt 2·75 × 106) appears to consist of two hexagonal discs apposed one to the other, each containing six identical subunits A located at the corners of the hexagon. Each subunit A is composed of three smaller structures B. On the basis of the size of the subunits B and data obtained in solution, it is concluded that two chlorohaems are contained in each subunit B, to give a total number of 72 haems per molecule. It is suggested that the co-operative interactions observed in the oxygen equilibrium arise within the subunits A, each of which contains 6 haems.

The properties and interactions of the isolated alpha- and beta-chains of human haemoglobin. IV. Immunological studies involving antibodies against the isolated chains.

(1) Antibodies against the isolated α - and β -chains of human haemoglobin produce precipitation and complement fixation with their antigens. (2) Both types of antibody give complement fixation with haemoglobin A, but do not cross-react with the opposite type of chain. (3) The behaviour of the anti α - and anti β -sera is not affected by the presence of p -mercuribenzoate in the immunizing antigen. (4) The reaction of the anti α -serum with free α -chains is the same as with α -chains in native haemoglobin; however, when the accompanying β -chains are replaced by those of another species in hybrids, or by γ -chains in foetal haemoglobin, the strength of the reaction of the anti α -serum is reduced in a specific way. (5) The reaction of anti β -serum with free β -chains is much stronger than when the β -chains are combined with α -chains in native haemoglobin; however, replacement of the α -chains by those of another species in hybrids, or by mutant α -chains in haemoglobin Burlington, has no effect. (6) After anti β -serum has been absorbed by haemoglobin A, it is still capable of giving complement fixation with free β -chains. This shows the presence of an anti β -antibody specific to the free β -chains. (7) The anti α - and anti β -sera are both highly sensitive to even one residue substitution in the chains against which they are directed.

The dissociation and recombination of subunits of human, horse, and canine hemoglobin

Experiments on the hybridization of various forms of human hemoglobin and of hemoglobins of different species at acid and alkaline pH raise a number of questions. In this paper we report experiments on the attempted hybridization of human and canine, and human and horse, hemoglobins by two hitherto untried procedures. In one of these the two hemoglobins were kept together at neutral pH and high ionic strength ( I = 3), where dissociation into half molecules may be assumed to occur, and then dialysed back to an ionic strength of 0·1. In the other a mixture of the globins of the two species, under conditions where these are dissociated either into half or quarter molecules, was combined with heme to obtain the reconstituted hemoglobins. In both cases the results were negative, i.e. no hybrid forms were produced.