Structural investigation of the complexation properties between horse spleen apoferritin and metalloporphyrins.

Crystallographic studies of L-chain horse spleen apoferritin (HSF) co-crystallized with Pt-hematoporphyrin IX and Snprotoporphyrin IX have brought significant new insights into structure-function relationships in ferritins. Interactions of HSF with porphyrins are discussed. Structural results show that the nestling properties into HSF are dependent on the porphyrin moiety. (Only protoporphyrin IX significantly interacts with the protein, whereas hematoporphyrin IX does not.) These studies additionally point out the L-chain HSF ability to demetalate metalloporphyrins, a result which is of importance in looking at the iron storage properties of ferritins. In both compound investigated (whether the porphyrin reaches the binding site or not), the complexation appears to be concomitant with the extraction of the metal from the porphyrin. To analyze further the previous results, a three-dimensional alignment of ferritin sequences based on available, crystallographic coordinates, including the present structures, is given. It confirms a high degree of homology between these members of the ferritin family and thus allows us to emphasize observed structural differences: 1) unlike L-chain HSF, H-chain human ferritin presents no preformed binding site; and 2) despite the absence of axial ligands, and due to the demetalation, L-chain HSF is able to host protoporphyrin at a similar location to that naturally found in bacterioferritin.

Embolism detected by transoesophageal echocardiography during hip arthroplasty.

This case report demonstrates embolization of echogenic material detected by transoesophageal echocardiography during a cemented total hip arthroplasty in a 76-yr-old woman without patent foramen ovale. During the placement of the acetabular and femoral components, and during relocation of the hip joint, a "snow flurry" appearing in the right atrium was followed by several highly echogenic and mobile emboli of various sizes, some of them with a vermiform shape 1 to 5 cm long. At skin closure, echogenic material was seen in the right branch of the pulmonary artery adherent to the vascular wall of the bifurcation of the main pulmonary artery. No changes were observed in any haemodynamic variable monitored (heart rate, systemic and right atrial pressures). Also, no desaturation was detected by pulse oximetry and blood gases at the time of embolism and there was no decrease in PETCO2. This case report is in line with other studies which failed to show a haemodynamic impact of TEE detected emboli during THA.

Crystallization and preliminary X-ray analysis of an orthorhombic form of horse-spleen apoferritin.

Horse-spleen apofemtin crystallizes in two different space groups: cubic F432 and tetragonal P42(1)2 while its iron-containing analogue is known to present a cubic and an orthorhombic form. Up to now, only the structure of the cubic form has been fully investigated by X-ray diffraction, although some information concerning the molecular packing of the two other forms was deduced from analysis of X-ray photographs. While growing cubic crystals of horse-spleen apoferritin with Pt-mesoporphyrin IX, we obtained one crystal, with a diffraction limit of 2.4 A, belonging to the orthorhombic P2(1)2(1)2 space group, with unit-cell dimensions a = 181.6, b = 128.9, c = 128.9 A. The orientation of the non-crystallographic axes of the molecule was determined by self-rotation Patterson function and the structure was determined by the molecular-replacement method. The asymmetric unit consists of half an apoferritin molecule. Refinement of the structure is in progress, some preliminary results of the molecular packing are given.

Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH.

In previous studies it has been shown that reaction of crystalline horse spleen apoferritin with hemin leads to a protoporphyrin IX-apoferritin complex [Précigoux et al. (1994) Acta Crystallogr. D50, 739-743]. We show here the following. (i) Hemin binds to two classes of sites in horse spleen apoferritin at pH 8, each with a binding stoichiometry of 0.5 hemin/subunit; protoporphyrin IX also binds to horse spleen apoferritin with an apparent binding stoichiometry of 1 molecule of protoporphyrin IX/subunit. (ii) When Fe(III)-protoporphyrin IX binds to apoferritin, there is a pH-dependent loss of the metal ion, extremely slow at alkaline pH values (half-time of weeks) and much more rapid at acidic pH values (half-time of seconds below pH 5.0); maximum rates of demetallation are found at pH 4.0, and at lower pH values they decrease. (iii) Chemical modification of 11 carboxyl groups/subunit in horse spleen apoferritin does not affect hemin binding at alkaline pH values; however, it prevents hemin demetallation at acidic pH values. (iv) Hemin that has been demetallated at acidic pH values can be remetallated by increasing the pH; the rate of remetallation is greater at more alkaline pH values. (v) When around 20 atoms of iron/molecule are incorporated into horse spleen apoferritin and protoporphyrin IX is then bound, iron can subsequently be transferred to the porphyrin at pH 8.0. A mechanism is proposed to explain demetallation of heme, involving attack on the tetrapyrrole nitrogens of the protoporphyrin IX-Fe by protons derived from protein carboxylic acid groups and subsequent complexation of the iron by the corresponding carboxylates and binding of protoporphyrin IX to a preformed pocket in the inner surface of the apoferritin protein shell. The cluster of carboxylates involved is situated at the entrance to the pocket in which the protoporphyrin IX molecule is bound and has been previously identified as the site of iron incorporation into L-chain apoferritins. This appears to be the first example of iron removal and incorporation into porphyrins under relatively mild physiological conditions.