No abstract

4-triazole interact with methaemoglobin from Chironomus plumosus. In a weakly acidic environment all complexes show light absorption spectra of the parahaematin type with maxima near 535 and 415 mp. 2. The affinity of these ligands to Chironomus-methaemoglobin rises with increasing basic strength, with the exception of 3-amino-l,2,4-triazole. 3. The spectra of the N-alkylated imidazoleor triazole-complexes are pH-invariant, while those of the complexes with ligands containing a free NH-group shift their maxima to longer wavelengths in alkaline medium (about 542 and 417 mp). 4. This spectral change is attributed to the deprotonation of the NH-group of the bound ligands. The ionization constants of this reaction have been estimated. They demostrate a strong increase of the acidity of these ligands as a result of their interaction with the haemoprotein. 5. Furthermore, the ligands influence the ionization constants of some haemin-linked protein groups. 6. The results are an example of the changes of the electronic properties of low-molecular compounds (drugs etc.) interacting with specific receptors of biomacromolecules and vice versa.

I. By means of an analytical ultracentrifuge the molecular weight of purified methaemoglobin from Chironomus plumosus has been determined a t different pH values. Under weak acidic conditions (pH 5.2 to 7.0) the sedimentation constant amounts to 2.73 & 0.02 S and the diffusion constant varies from 7.5 to 8.5 F. The molecular weight was calculated as 32,000 & 1,500. 2.Outside of this pH-range the molecular weight decreases. I n alkaline solution (pH 9.2-10.0) the dimer molecule splits off reversibly into monomer subunits, with an average molecular weight of 14,600 4 : 300.3. I n the pH-range 7.5 to 9.0 and below 5.0 there exists a mixture of dimer and monomer molecules. The monomer-dimer-equilibrium is pH-dependent. pH,,, of this equilibrium coincides with the pH,,, of the spectrophotometrically estimated main ionization constant (pH,,, = 8.3). The deprotonation of a haemin-bound water molecule induces a change of the quarternary structure of the Chironomus-methaemoglobin-molecule.4. According to the frictional ratio and the Stoke-radius the monomer and the dimer MetHbmolecules have an asymmetrical shape. Assuming a rotation ellipsoid (prolate type) for the monomer form (pH > 9.2) the axes are about 24 and 12.6 A. Unfortunately no exact information on the environmental conditions are given in their paper. I n the meantime Braunitzer and Braun [S] published the amino acid composition of one Hb-fraction from Chironomus thummi. With nearly 125 amino acids for the a-and 127 for the 8-chain the authors found smaller subunits than in the vertebrate-Hbs. Following studies on the X-ray structure of the crystallized Chironomus-Hb [9] our results provide some further information on several molecular properties of this Hb in solution. EXPERIMENTAL PROCEDURE MaterialPreparation of the crude Chironomus-MetHb solution was carried out by a method described by us earlier [i]. The crude material was purified chromatographically on Sephadex G 75 or G 100 (Pharmacia, Uppsala) and Epidex A 10 or B 10 (VKB Sernmwerk Bernburg) respectively, using a 0.05 M NaH,PO,/NaCl-buffer pH 7.4 and I = 0.1. Ch' zronomus-Hb is readily autoxidable. It appears in two oxidized fractions a t the end of the preparation. The first one shows the normal absorption spectrum of the Chironomzcs-MetHb, which we have published earlier [l]. This fraction was used for the further investigations. The second fraction contained denaturated MetHb-products and was rejected. MethodsSedimentation measurements were carried out with an analytical ultracentrifuge, model G 110 from MOM Budapest, with schlieren optics. Additionally we used a SCHOTT-filter OG 3. All the experiments were performed at 50,000 rev./min and in the temperature range 1.9.5 to 22".

In alkaline aqueous solution, the sedimentation coefficients of DeoxyHb, HbO2 and HbCO of Lampetra fluviatilis L. are 1.9 f 0.1 S. This value corresponds to a molecular weight of about 17,000, i.e. the value of a single haem polypeptide chain. In weak acidic conditions, both the non-liganded DeoxyHb and the liganded forms, HbO2 and HbCO, associate to form dimers and oligomers. The monomer-oligomer transitions of these compounds take place at different pH values: DeoxyHb pHO.5 ?. 6.7; HbO2 and HbCO pHO.5 %I 5.9. With respect to the association modus, the equilibrium: 4 Hb * 2 Hb2-b4 may be preferred.

No abstract

No abstract

1. In alkaline aqueous solution (pH > 10) protohaemin forms a green coloured pyridine complex containing two ligands per haemin dimer. The thermodynamics of this reaction is characterized by a considerable entropy changc due to hydrophobic interactions between both partners.2. Similar haemin complexes are formed by N-alkyl-pyridinium halides. The absorption spectra are nearly identical to those of the corresponding pyridine complcxes although there is no possibility of interaction with the positively chargcd haemm iron. Pyridinium cations dcvelop a higher affinity towards haemin than the neutral pyridines. This bchaviour is attributed to electrostatic attractions between the propionylic groups of porphyrin and the positively charged pyridinium salts.3. The introduction of methyl groups intensifies the haemin complex stability both of the pyridines and the pyridinium salts. In order of increasing methylation of the ligands the reaction entropy incrcascs. 4.Following the substitution of pyridine in position 4 by a cyano group or following the transition from N-alkyl-pyridinium halides to N-alkyl-quinolinium halides the affinity towards haemin increases and the thermodynamics show a decrease of the reaction enthalpy whilst the entropy of thc system changes only little. For this behaviour n-donor-acceptor interactions should be rcsponsible.5. The results demonstrate the contribution of several types of chemical bond in the reaction of protohaemin with ligands. Apart from the well investigated coordinative linkage to the iron atom, n-donor-acceptor and hydrophobic interactions between ligands and the porphyrin system as well as ionic bonds between the propionylic groups of the porphyrin and positively charged ligands contribute to their haemin complex formation. 6 . The significance of the results is discussed with regard to the haemin-protein interaction in haemoproteins.

No abstract