Irina Shin

A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.

Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is “native‐like,” and inactivation can be reversed by exposure to reduced glutathione. The allicin‐modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t1/2 ≃ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine‐N‐oxide, glycerol, and sucrose, and the divalent cations, Ca2+, Mg2+, and Mn2+ can prevent this transition of the native‐like state for >24 h at room temperature. Trimethylamine‐N‐oxide and Mg2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native‐like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4‐trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first‐order rate constant for thermal inactivation at 45°C, of single molecules entrapped within the reverse micelles (0.031 min−1), was higher than in aqueous solution (0.007 min−1) or in the presence of normal micelles (0.020 min−1). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first‐order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53°C, were 0.317 and 0.342 min−1, respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.

Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well‐known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold‐type I family of pyridoxal‐5′‐phosphate‐dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a SS bridge located near the C‐terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate‐cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored SH reagent N‐(4‐dimethylamino‐3,5‐dinitrophenyl) maleimide yielded chromophore‐bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free SH groups. This allowed the oriented conjugation of alliinase, via the SH groups, with low‐ or high‐molecular‐weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.

Therapy of Murine Pulmonary Aspergillosis with Antibody-Alliinase Conjugates and Alliin

ABSTRACT Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Stabilization of Alliinase from Garlic by Osmolytes and the Mannose-Specific Lectin ASAI

Objectives: Alliinase is a pyridoxal-5’-phosphate (PLP)-dependent enzyme responsible for the production of diallyl thiosulfinate (allicin), the biologically active component of garlic, from alliin. The use of allicin for treatment of various diseases has been proposed but it is very unstable in the blood stream. This difficulty can be overcome by administration of alliin, together with a conjugate of alliinase directed towards the target cells. This, in turn requires a stable and active form of the enzyme. In this study we evaluate the stability of alliinase itself, in the presence and absence of osmolytes, as well as that of its catalytically active complex with a mannose-specific lectin, ASAI (Allium sativum agglutinin I), also presents in garlic. Methods: Alliinase, and ASAI were both purified from garlic cloves. Thermal stability of alliinase itself, and of its complexes with PLP and ASAI, in the presence and absence of osmolytes, was analyzed by monitoring enzymic activity, and using DSC (differential scanning calorimetry). Key findings: PLP exerts only a minor influence on alliinase structure and stablity. But both osmolytes and ASAI stabilize the enzyme considerably. Conclusions: The principle finding is that ASAI greatly stabilizes alliinase. Thus, the lectin-enzyme complex, which can be lyophilized and stored until used, provides an effective formulation of alliinase for generation of allicin from alliin in vivo.

Membrane-Promoted Unfolding of Torpedo Californica and Bungarus Fasciatus Acetylcholinesterase

The kinetics and thermodynamics of interaction of proteins with the lipid bilayer are important for understanding their mode(s) of insertion into and translocation across biological membranes. These, in turn, may be relevant to the mechanisms of both cotranslational and posttranslational cellular traffic of proteins. Involvement of the lipid bilayer may also be envisaged in the so-called ‘conformational’ diseases, such as prion diseases (e. g. Creutzfeld-Jacob disease) and amyloid diseases (e. g. Alzheimer’s disease). In some such diseases, at least part of the protein is correctly folded in a ‘native’ conformation, and the disease condition arises from subsequent conformational changes leading to aggregation and deposition of the protein.

Studies on Partially Unfolded States of Torpedo californica Acetylcholinesterase

Chemical modification, by a repertoire of thiol reagents, of the non-conserved Cys231 residue of Torpedo californica acetylcholinesterase (AChE), results in inactivation, even though Cys231 is not involved in catalysis (Steinberg et al., 1990; Dolginova et al., 1992; Silman et al., 1992; Salih et al., 1993). Modification by disulfides and alkylating agents produces partial unfolding of native AChE (N) to a compact state resembling a molten globule (MG). The MG is a collapsed structure possessing much of the secondary structure of the fully folded native protein, but devoid of tertiary structure (Kuwajima, 1989; Ptitsyn, 1992); it is currently believed to serve as a folding intermediate en route from the nascent polypeptide chain, generated on the ribosome, to the fully folded native protein (Kim and Baldwin, 1990; Gething and Sambrook, 1992). This structural assignment for the species produced by chemical modification of Torpedo AChE was based upon spectroscopic evidence, including CD, intrinsic fluorescence and binding of ANS, upon hydrodynamic measurements, including sucrose gradient centrifugation and quasielastic light scattering, and upon enhanced sensitivity to proteolysis (Dolginova et al., 1992). Although modification by disulfides can be rapidly reversed by exposure to reduced glutathione (GSH), the native (N) conformation is not restored, and no catalytic activity is recovered. AChE so demodified is a partially unfolded species whose physicochemical characteristics are virtually identical to those of the modified enzyme, and sucrose gradient centrifugation reveals it to be stable for many hours without aggregating.