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1.
Chemistry ; 30(33): e202400082, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38628039

RESUMO

Fagopyrins are phenantroperylenequinones present in the flowers of Fagopyrum esculentum (buckwheat) endowed with photodynamic activity. It has been reported that fagopyrin extracts actually contain a complex mixture of closely related compounds, differing only on the nature of the perylenequinone substituents. We report our systematic and detailed study on the chemical composition of fagopyrin extracts by a combination of preparative and analytical techniques. The combined use of 1H-NMR and CD spectroscopy was found to be particularly suited to fully characterize all stereochemical aspects of the extracted fagopyrins. For the first time nine isomers have been structurally characterized and their stereochemistry fully elucidated. The presence of two different heterocyclic ring substituents, two stereogenic centers and the inherent axial chirality of the aromatic system provides a complex stereochemical relationships among isomers, thus giving account of the high level of molecular multiplicity found in the extract.


Assuntos
Dicroísmo Circular , Fagopyrum , Flores , Fagopyrum/química , Flores/química , Estereoisomerismo , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Estrutura Molecular , Extratos Vegetais/química , Quinonas
2.
Phytochemistry ; 69(9): 1820-5, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18474386

RESUMO

The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (Mr 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2+/-0.4 nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman-Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems.


Assuntos
Lupinus/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Quimotripsina/antagonistas & inibidores , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Cinética , Lupinus/química , Dados de Sequência Molecular , Sementes/química , Sementes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Titulometria , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificação
3.
FEBS J ; 273(17): 4024-39, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16889634

RESUMO

Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 microM-1.s-1 vs. 0.002 microM-1.s-1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal beta-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.


Assuntos
Lens (Planta)/química , Sementes/química , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Lens (Planta)/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sementes/metabolismo , Soluções , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificação
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