Paulo Boschcov

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N α-Boc or-Fmoc derivative of glutamic acid with the α-carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the α-amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum

1 Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. 2 The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 μM), but pre‐incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. 3 The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. 4 Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. 5 Des‐Arg9‐bradykinin, an analogue specific for B1‐receptors, was much less active than bradykinin, and elicited only a contractile response. 6 Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. 7 Our results support the hypothesis that the relaxant and contractile components of the rat duodenums response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.

Intramolecularly quenched fluorogenic peptide substrates for human renin

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.

Autoantibody Response to Chromatographic Fractions from Oxidized LDL in Unstable Angina Patients and Healthy Controls

Levels of autoantibodies to oxidized low‐density lipoprotein (oxLDL) have been correlated to atherosclerosis; however, contradictory results have been shown. To better understand the role of autoantibodies to oxLDL in atherogenesis, and their potential to predict risk of developing coronary artery disease we investigated the antibody response of unstable angina (UA) patients and healthy controls against chromatographic separated fractions of oxLDL. Five major peaks were detected after chromatographic separation of oxLDL and 10 fractions were collected. Surprisingly, when the response to high molecular weight fractions was analysed, we observed a significant increase in the levels of autoantibodies in controls compared to UA. In contrast, when the autoantibody response to intermediate and low molecular weight fractions was analysed, we observed that the UA group showed consistently higher levels compared with controls. Our data demonstrates that within oxLDL there are major fractions that can be recognized by autoantibodies from either UA patients or healthy individuals, and that the use of total oxLDL as an antigen pool may mask the presence of some antigenic molecules and their corresponding antibodies. Further studies are needed, but the analysis of antibody profiles may indeed open up a novel approach for evaluation and prevention against atherosclerosis.

Ionization constants and thermodynamic parameters of histidine and derivatives

Abstract The pKa values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pKa values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pKa in histidine and its studied derivatives. The Nτ-H tautomer is favored over the Nπ-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.

Macrophages take up triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL: An investigation on the role of natural chylomicrons in atherosclerosis

Chylomicrons play a role in atherosclerosis, however, because the mechanisms involved in the cell uptake of these particles are not fully understood, investigations were carried out using a radioactively labeled protein‐free triacylglycerol‐rich emulsion incubated with peritoneal macrophages obtained from normal and apoE‐knockout mice. Experiments were done in the presence of substances that inhibit several endocytic processes: EDTA for low density lipoprotein receptor, fucoidan for scavenger receptor, cytochalasin B for phagocytosis, and a lipopolysaccharide for lipoprotein lipase. In addition, triacylglycerol‐rich emulsions were also prepared in the presence of native or modified radioactively labeled low density lipoprotein particles that are known to accumulate in the arterial intima. Probucol was also used to prevent the possible role played by an antioxidant in triacylglycerol‐rich emulsion uptake. We have shown that triacylglycerol‐rich emulsion alone is taken up by a coated‐pit‐dependent mechanism, mediated by macrophage secretion of apolipoprotein E. Furthermore, native, aggregated, acetylated, and moderately macrophage‐oxidized low density lipoprotein stimulate the uptake of a triacylglycerol‐rich emulsion through several mechanisms such as an actin‐dependent pathway, scavenger receptors, and lipolysis mediated by lipoprotein lipase. On the other hand, in spite of the interaction of low density lipoprotein forms with a triacylglycerol‐rich emulsion, the cellular triacylglycerol‐rich emulsion uptake is impaired by copper‐oxidized low density lipoprotein, possibly due to its diminished affinity towards lipoprotein lipase. We have also shown that macrophages take up aggregated low density lipoprotein better than the acetylated or oxidized forms of low density lipoprotein. J. Cell. Biochem. 84: 309–323, 2002.

Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione

Theortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. In fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH2, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH2 or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH3)2, Abz-Pro-Leu-Gly-NH2 or Abz-pyrrolidine. For all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. In conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.

Reactivity with p-nitrophenyl acetate and interaction between the amino and imidazole groups of histidine and related compounds

Abstract The study of the reaction of p-nitrophenyl acetate (PNPA) with histidine and certain derivatives showed that the species in which the amino group is unprotonated (R(NH2)Im) react with second-order rate constants ( k 2 am ) that are higher than predicted by a Bronsted relation for a series of neutral amino acids. The reason for this behavior was investigated through an analysis of the kinetics of the reaction of PNPA with these compounds in order to assess the reactivities of the amino and imidazole groups in the two species R(NH 3 + ) Im and R(NH 2 ) Im . The rate constant for the reaction with the imidazole group ( k 2 im ) of Nπ-methyl histidine agrees with the value predicted by a Bronsted relation obtained from a series of model imidazole compounds. Nτ-Methyl histidine, however, is unreactive, indicating that Nτ is the reactive nitrogen in the imidazole ring of histidine. The k 2 im values found for histidine, histidine methyl ester, and Nα-dimethyl histidine are lower than predicted by the Bronsted relation. This behavior was found to be due to low reactivity of the R(NH 3 + ) Im species, in contrast with the normal reactivity of R(NH 2 ) Im . The evidence presented suggests that the lower reactivity of R(NH 3 + ) Im is due to an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring, which displaces the tautomeric equilibrium toward the unreactive Nτ-H form. The higher reactivity of the imidazole group in the species R(NH2)Im, relative to that in R(NH 3 + ) Im , is responsible for the observed high k 2 am values for histidine, for histidine methyl ester, for Nτ-methyl histidine, and for Nα-dimethyl histidine, in contrast with the normal k 2 am value found for Nτ-methyl histidine. The conclusions from this study of histidine and its derivatives support the proposal of an interaction between the protonated N-terminal amino group and the imidazole ring of His6 in the octapeptide hormone angiotensin.

Antihypertensive therapy increases natural immunity response in hypertensive patients

AIMS The aim of this work was to evaluate the effects of treatment of hypertension on the autoantibodies to apolipoprotein B-derived peptides (anti-ApoB-D peptide Abs) response, inflammation markers and vascular function. MAIN METHODS Eighty-eight patients with hypertension (stage 1 or 2) were recruited and advised to receive perindopril (4mg), hydrochlorothiazide (25mg), or indapamide (1.5mg) for 12weeks in a blinded fashion. Office and 24-h ambulatory blood pressure monitoring (24h ABPM), flow-mediated dilatation (FMD), nitrate-induced dilatation (NID), titers of IgG and IgM anti-ApoB-D peptide Abs, hsCRP, and interleukins (IL-8 and IL-10) were evaluated at baseline and 12weeks after therapies. KEY FINDINGS All treatments reduced office BP, and improved FMD (P<0.05 vs. baseline). The NID was improved only in the perindopril arm (P<0.05 vs. baseline). The 24h-ABPM was reduced with perindopril and hydrochlorothiazide therapies (P<0.05 vs. baseline), but not with indapamide, and this effect was followed by increase in titers of IgM Anti-ApoB-D peptide Abs (P<0.05 vs. baseline), without modifications in titers IgG Anti-ApoB-D peptide Abs and interleukins. Multivariable regression analysis has shown that change in the titers of IgM anti-ApoB-D peptide was associated with the changes in FMD (β -0.347; P<0.05). SIGNIFICANCE These findings shed light to a possible modulator effect of the antihypertensive therapy on the natural immunity responses and vascular function.