Anne d’Albis

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.

Myosin polymorphism in muscles has been studied by a variety of electrophoretic techniques, in non-dissociating and in dissociating conditions. The analysis of myosin isozymes in the native state was achieved in pyrophosphate buffer and required only minute amounts of protein; identical results were obtained with purified or crudely extracted myosin. The determination of the subunit content of each isozyme was done in the presence of sodium dodecyl sulphate or urea for light chain, and in a phenol, acetic acid and urea system for heavy chain screening. Electrophoresis in non-dissociating conditions has led to the separation of up to a dozen of myosin isozymes, differing in mobilities by as much as 30%. Muscle specificity of myosin was clearly established. Apart from a few exceptions, all the muscles tested were shown to contain more than one myosin species; fast-twitch muscles for instance all contained the same three isozymes, but in variable ratios. Class specificity of myosin appeared related to the relative proportions of isozymes in a given muscle. A second electrophoresis in dissociating solvents of the myosin bands first resolved in pyrophosphate buffer has then allowed a further characterization of the various isozymes. The differences in mobilities observed in the native state were shown to come either from the light chains, or from the heavy chains, or from both. The first case was illustrated by the three species present in fast muscles, which were shown to correspond to three alkali light-chain isozymes, the heterodimer representing in some instances up to 40% of the total. Next to light-chain muscle type specificity, electrophoresis in the phenol, acetic acid, urea system has led to the detection of differences in the heavy chains of fast, slow and cardiac myosins. The application of these various electrophoretic techniques to the analysis of the modification of myosin isozymes during development or in pathology studies can be considered.

The regeneration of adult rat and mouse slow (soleus) and fast (sternomastoid) muscles was examined after the degeneration of myofibers had been achieved by a snake venom cardiotoxin, under experimental conditions devised to spare as far as possible the satellite cells, the nerves, and the blood vessels of the muscles. Three days after the injury, no myosin was detectable in selected portions of the muscles. New myosins of embryonic, neonatal, and adult types started to be synthesized during the following two days. Adult myosins thus appeared more precociously than in development, which implies that the synthesis of myosin isoforms during regeneration does not entirely 'recapitulate' the sequence of myosin transitions observed during normal development. Two weeks after the injury, the isomyosin electrophoretic pattern displayed by regenerated muscles was already the same as that of control muscles; the normal adult pattern was therefore expressed more rapidly in regenerating than in developing muscles. Except for the synthesis of the slow isoform which was generally inhibited in denervated muscles, the same types of myosins were expressed during the early stages of regeneration in denervated as in innervated muscles; long-term denervation prevented however the qualitative and quantitative recovery of the normal myosin pattern.