Alberto Bertollini

The hydrolysis of α-CBZ-l-lysine-p-nitrophenyl ester by two forms of human urokinase

Abstract The catalytic properties of human urokinase have been investigated using a synthetic chromogenic substrate; α-CBZ- l -lysine-p-nitrophenyl ester (ZLNP). The enzymatic assay based on the rate of hydrolysis of ZLNP offers several advantages over other methods currently employed in different laboratories. The steady state parameters of the two purified forms of human urokinase, which differ in molecular weight (33,000 and 54,000 daltons), have been determined over the pH range 5.2–7.8, and found to be indistinguishable.

Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to −3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).

Interaction of inositol hexakisphosphate with liganded ferrous human hemoglobin. Direct evidence for two functionally operative binding sites

Inositol hexakisphosphate (InsP6) binding to the oxygenated, carbonylated and nitrosylated derivatives of ferrous human hemoglobin (HbO2, HbCO and HbNO, respectively) has been measured at pH 7.0 (0.1 M Bis-Tris buffer, 0.1 M NaCl) and 20 degrees C. The observations indicate the presence of two InsP6 binding sites per tetramer in all the heme liganded hemoglobin derivatives, with different affinities for the polyphosphate. For each binding site, InsP6 interacts with similar affinity constants to HbO2, HbCO and HbNO. Such a finding indicates that different heme ligands do not alter significantly the stereochemistry of the polyphosphate binding cleft. This behaviour seems to indicate that, even though different heme ligands are likely to affect the tertiary conformation of the subunit in a different fashion, the perturbation does not seem to be transmitted to the quaternary arrangement of the whole macromolecule, and, thus, to the InsP6 binding site.

Primary specificity of ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom.

Values of steady-state and pre-steady-state parameters for the hydrolysis of ZArgONp and ZLysONp catalysed by ancrod, the coagulating serine proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom, have been determined, between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C, and analysed in parallel with those of bovine alpha-thrombin and porcine pancreatic beta-kallikrein-B. In addition to the well-known coagulating behaviour, ancrod also shows catalytic properties, in the hydrolysis of ZArgONp and ZLysONp, reminiscent of those of porcine pancreatic beta-kallikrein-B.

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between beta-chains along the dyad-axis is sterically hindered in HbA1c by the presence of the two glucose residues covalently bound to the N-termini of beta-chains, this finding being in agreement with the reduced effect of polyanions on HbA1c spectral and ligand-binding properties.

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.

899 — A potentiometric study on the redox properties of hemoglobin from Camelus dromedarius

Abstract A potentiometric study of the redox properties of hemoglobin from Camelus dromedarius has been performed, with the aim of having a better insight in the structure-function relationships of this protein; data on human hemoglobin have been included for comparative purposes. Analysis of the experimental data has shown: 1. the existence of intermediate conformers (neither R nor T ) in the oxidized form of dromedary hemoglobin; 2. the amino acid groups involved in the oxidation Bohr effect of dromedary and human hemoglobins are likely to be the same; thus, only invariant amino acid residues seem to be involved in this effect.

Catalytic properties of Ancrod, the thrombin-like proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom

Kinetics for the hydrolysis of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids catalyzed by Ancrod were determined between pH 5 and 10 (I = 0.1 M) at 21 +/- 0.5 degrees C; the results are consistent with the minimum three-step mechanism: (formula: see text) For all substrates examined, the pH profiles of kcat and/or kcat/Km reflect the ionization of two groups with pKa values ranging between 6.9 and 7.2, and 9.3 and 9.6 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); at variance, values of Km are pH-independent. Moreover, the formation of the E X S complexes may be regarded as a pseudo-equilibrium process, and the acylation step (k + 2) is always rate-limiting in catalysis. Among p-nitrophenyl esters examined, ZArgONp shows the most favourable kinetic parameters and may be the substrate of choice for Ancrod, in that it allows the determination of the enzyme concentration as low as 1 X 10(-9) M (approximately equal to 0.1 Ancrod units/ml), at the optimum pH value (approximately equal to 8). The catalytic behaviour of Ancrod is compared to that of serine proteinases acting on cationic and non-cationic substrates; differences in kinetics, which refer to a lower enzyme:substrate affinity, may be related to a higher rigidity, lower hydrophobicity and/or adverse steric hindrance of the S1 subsite of Ancrod.

Iron site structure of two irreversible hemichromes from human hemoglobin, untreated and oxidized to sulfoxide at MetD6(55) β

The Fe K-edge X-ray absorption near-edge structure (XANES) spectra of two irreversible human hemichromes, spontaneously formed from HbA and HbMetSO (a hemoglobin derivative, where MetD6(55)beta has been previously oxidized to sulfoxide by chloramine T) were determined. The results show that the hemichrome from HbMetSO is characterized by the distal histidyl imidazole moved within the bonding distance of the heme iron. Such structure is different from that of the hemichrome spontaneously produced from native human hemoglobin, which probably has a hydroxide group as sixth heme ligand.

A Potential, Intracellular Trigger for Removal of Senescent Erythrocyte: Hemoglobin with Methionine Beta (55) D6 Oxidized to the Sulfoxide Derivative

Most of the prevalent theories of aging are working hypotheses designed to measure and explain various age-related changes. The basic assumption of the theories commonly referred to as stochastic is that cellular aging may be due to cumulative and deteriorative effects of random events, such as the chemical damages due to free radicals (1). In recent years (since the discovery of superoxide dismutase), oxygen radicals have been invoked to favor debilitating processes, including aging of molecules and cells (1,2). Although oxidation of thioesther groups has long been known in biochemistry, the importance of methyonyl residues as possible targets of oxygen radicals has only recently been recognized (3). Methionyl residues in proteins and peptides can readily be oxidized also in vitro to the sulfoxide derivative by suitable chemical agents, under mild conditions, which do not modify other residues except exposed sulfhydryl groups. Therefore, in view of the effect of the oxidation of methionine in several biological processes (such as aging), it seemed of interest to investigate the functional properties of human hemoglobin with some methionyl groups oxidized by chloramine T.