G. H. Beaven

The interaction of haemin with human serum albumin has been reexamined. The absorption spectrum of the bound haem is identical with that of uncomplexed monomeric haemin in solution, and it is suggested, on the basis of an interaction of albumin with iron‐free protoporphyrin IX, that the iron is not implicated in the interaction with the protein. A ferric cyanide derivative, and a ferrous haem derivative of methaemalbumin can be recognised, but not azide or fluoride derivatives. The bound haemin gives rise to extrinsic Cotton effects, which are different in detail in the derivatives, and in the complex with protoporphyrin IX Spectrophotometric titrations show that there is one strong binding site for haemin and several weaker sites. The latter are associated with optical activity opposite in sign to that of the strong complex. The binding profiles are little affected by pH over a wide range, by ionic strength or by the presence of 40% (v/v) dimethylsulphoxide/water, in which the free haemin is monomeric. The binding of haemin to albumin has been followed by spectrophotometry, circular dichroism and fluorescence. The binding of haemin quenches the protein fluorescence, which progressively changes in character from tryptophan to tyrosine type. The haem at the primary binding site thus strongly quenches the tryptophan specifically. From fluorescence titrations at a range of protein concentrations, binding isotherms were constructed, and gave an association constant for the strong site of 50 μM −1 . From binding isotherms based on absorption measurements we can infer the existence of at least four sites with much lower binding constants. A long‐chain fatty acid anion was found to compete with haemin only for the weaker binding sites, so that binding beyond one mole per mole of protein can be essentially eliminated. The open‐chain tetrapyrrole, bilirubin, was found, in agreement with earlier work, not to compete with haemin, as regards the strongest binding sites of either ligand. Between the weaker sites, however, evidence of competition was obtained.

The interaction of bilirubin with de‐fatted human serum albumin has been studied by circular dichroism and spectrofluorimetry. From the dependence of the induced circular dichroism of the bilirubin on the ratio of protein to bilirubin, it is shown that in the presence of salt (0.5 M sodium chloride) there are two binding sites with markedly different optical characteristics. From an analysis of the profiles of ellipticity as a function of protein: bilirubin ratio, association constants of 7 and 0.3 μM −1 at pH 8.5 were derived. At low salt concentrations qualitatively similar binding profiles are obtained with a marked maximum of ellipticity at a protein: bilirubin ratio of about 1. These cannot however be adequately accounted for with only two binding sites. The addition of a third site allows the data to be fitted over the entire concentration range at all wavelengths tested, with association constants falling in the range 10–0.01 μM −1 . When bilirubin is bound to albumin, the fluorescence of the protein is quenched and that of the bilirubin is enhanced. The Fluorescence characteristics of the binding sites are shown to be different: the weaker sites, which have lower fluorescence, but higher circular dichroism than the stronger, are relatively much more labile to salt. The pH‐dependence of the optical properties reveals the presence of a state defined by a plateau at between pH 8 and 9, another by a plateau centred at about pH 6.5, and another, the appearance of which is associated with the N–F transition of the protein, and which exists only within an extremely narrow pH range, at about pH 4. The pH at which the minimum occurs undergoes a large shift with salt concentration. This state is characterised by an extremely large circular dichroism (effective molar ellipticity ‐5 × 10 5 deg. cm 2 · dmol −1 ), opposite in sign to the system of Cotton effects observed at higher pH. This complex shows no fluorescence enhancement. The system shows large hysteresis, satisfactory results being obtained only when the complex is first formed by equilibration at pH 8–9 at low ionic strength. Under other conditions the self‐association of the bilirubin evidently prevents the attainment of equilibrium, and vitiates any attempts at analysis of the binding equilibria. The competition of oleate ions with the bilirubin binding sites has been studied. Circular dichroism, under physiological conditions, of human serum containing bilirubin shows that the binding of the bilirubin by albumin alone at the corresponding ionic strength and pH defines the state of the ligand in the plasma.

Glucagon in acid solution aggregates to produce gels and ultimately fibrils of β‐chains. The kinetics of the aggregation have been followed by viscometry, and are found to exhibit a sigmoidal profile with a long lag phase, associated with the formation of nuclei for polymerisation. This has been demonstrated by the elimination of the lag when fresh solutions are seeded with a preformed glucagon gel. The aggregation is promoted by salt, increased pH (within the acid range of solubility) and increased temperature up to 30°. The addition of 5% of dioxan completely inhibits the aggregation. The interaction has evidently a large hydrophobic driving force, and is opposed by coulombic repulsion. The latter effect can be minimised by acetylation of the amino groups, when the polymerisation is greatly accelerated. This also occurs when only the terminal amino group is blocked by carbamylation. By contrast esterification of the carboxyl groups completely prevents aggregation. At the working pH this does not involve a change in charge, and thus appears to be a specific steric effect. Optical rotatory dispersion shows no conformational difference between native glucagon and the derivatives. The transition from a gel to a fibrillar form is promoted by salt and increased temperature. A possible explanation for the appearance of the fibrillar form in the electron microscope is that it contains tubular structures 50–70 Å in diameter, with about 40 extended chains around the circumference lying in antiparallel manner along the axis.