a b s t r a c tHuman serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k off ) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k off value increases from (1.4 ± 0.2) Â 10 À4 s À1 , in the absence of the drug, to (9.5 ± 1.2) Â 10 À3 s À1 , in the presence of 1.0 Â 10 À2 M ibuprofen, at pH 7.0 and 10.0°C. From the dependence of k off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K 1 = (3.1 ± 0.4) Â 10 À7 M, K 2 = (1.7 ± 0.2) Â 10 À4 M, and K 3 = (2.2 ± 0.2) Â 10 À3 M) were determined. The K 3 value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) Â 10 À3 M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow's site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.

Peptide-based metalloprotein models represent useful systems to help understand how metalloproteins can support different functions, by the use of similar metal ion cofactors. In order to shed light on the role of the protein matrix in modulating the heme properties, we developed new models: mimochromes. They are pseudo-C(2) symmetric systems, composed of two helical peptides covalently linked to the deuteroporphyrin. The use of C(2) symmetry is particularly advantageous, because it simplifies the design, synthesis and characterization. However, it leaves the problem of possible diastereomeric forms. In the cobalt complex of the first derivative, mimochrome I, Lambda and Delta isomers were indeed experimentally observed. All the insights derived from the Co(III)-mimochrome I structure were used to obtain a re-designed molecule, mimochrome IV. The spectroscopic characterization of the iron and cobalt derivatives suggested the presence of the Lambda isomer as unique species. The NMR solution structure of the diamagnetic Co(III)-mimochrome IV confirmed the ability of the molecule to adopt a unique topology, and revealed the peptide chains to be in helical conformation, as designed. The insertion of intramolecular, inter-chain interactions was successful in favoring the formation of one of the two possible diastereomers. The stereochemically stable structure of mimochrome IV provides an attractive model for modulating the redox potential of the heme, by simple changing the peptide chain composition around the heme.

Neutrophil collagenase MMP-81 is a Zn 2ϩ metallo-endopeptidase, which is able to cleave native triple-helical collagen I (1-3) at a specific peptide bond between Gly 775 and the residue at position 776 (which can be either Leu or Ile), leading to the formation of one-quarter and three-quarter fragments (4). MMP-8 is predominantly expressed by neutrophil precursors (5, 6), but very recently it has been demonstrated that it is also expressed in human articular chondrocytes (7) and that it might also be responsible for aggrecanase activity (8, 9). MMP-8 is expressed initially as a proenzyme and stored in the specific granules of neutrophils (10). The activation of MMP-8 can be accomplished either by mercurials, oxidative processes, and proteinases (11-14) thus removing a cysteinyl residue, which coordinates the Zn 2ϩ atom, rendering the enzyme inactive. In particular, it was observed that cathepsin G, a neutrophil serine proteinase, and stromelysin MMP-3 activate proMMP-8 through two different processes, the first one cleaving the Phe 79 -Met 80 peptide bond (11), whereas MMP-3 activates through the cleavage of the Gly 78 -Phe 79 peptide bond (15). Therefore, the resulting active MMP-8 displays two different N termini (i.e. Met 80 if activated by cathepsin G and Phe 79 if activated by MMP-3), and it has been shown that these two forms display meaningful differences in the catalytic activity toward synthetic substrates or inhibitors (16,17). The final active enzyme is made of a catalytic domain, where the Zn 2ϩ atom coordinated to 3 histidyl residues is located, and a hemopexin-like domain, which is connected to the catalytic domain through a linker region (18,19).Collagen I is one of the major components of the extracellular matrix for most tissues, such as skin, tendon, blood vessels, cartilage, bones, and basal laminae (20). It displays a triplehelical structural arrangement (21), which is kept by most of the molecule during the first cleavage event by MMP-8 (22), even though three-dimensional structures of collagenases indicate that the substrate binding cleft appears too narrow to accommodate a triple-helical collagen molecule. The two sets of data are not necessarily in contradiction, because a partial unwinding of collagen may be envisaged during the enzymatic action (23-25) while keeping the overall three-dimensional structure of the whole collagen molecule.Several studies have shown that, although the actual enzymatic action of collagenases occurs in the catalytic domain, the hemopexin-like domain plays an important role, because its * This work was supported in part by funds from the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST COFIN MM03185591 to M. C.) and the Deutsche Forschungsgemeinschaft, Bonn, Grants SFB 549 and SFB A5 (to H.T.) and Project Ts 8/35 (to H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely ...

The effect of lactate on O 2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O 2 affinity (i.e. an increase of P 50 ) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O 2 affinity appears to differ for sperm whale and horse heart Mb, ␦logP 50 being Ϸ1.0 and Ϸ0.4, respectively. From the kinetic viewpoint, the variation of O 2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.

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