Talia Miron

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 μM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 μM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G1/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

The mode of adsorption of proteins to aliphatic and aromatic amines coupled to cyanogen bromide-activated agarose

1. 1. Nonspecific adsorption of proteins to substituted agarose is a serious interference in affinity chromatography. Coupling of alkyl- or arylamines to CNBr-activated agarose, the most frequent technique of preparing affinity adsorbents with agarose, results in the formation of strong ion exchangers with an apparent pK of about 10 for a basic amidine nitrogen. Coupling of analogous hydrazides gives essentially uncharged agarose derivatives at physiological pH. 2. 2. α-Lactalbumin and ovalbumin were found to bind tightly to alkylagaroses with hydrocarbon chains of 4–8 carbon atoms. The same proteins were not adsorbed by the corresponding alkyl hydrazide derivatives of agarose. Bovine serum albumin adsorbed to both, amine- as well as hydrazide-substituted alkyl-agaroses. 3. 3. Leucine aminopeptidase showed no affinity for alkyl-agaroses but was bound strongly to tyramine-agarose with partial inactivation of the enzyme. Leucine aminopeptidase was not adsorbed by the hydrazide analogue of tyramine-agarose, the 4-hydroxyphenylacetyl hydrazide derivative. A detergent-like mode of action is assumed for alkyl- or arylamines coupled to activated agarose.

A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters.

Abstract A quantitative spectrophotometric assay for N-hydroxysuccinimide and its esters based on the formation of a chromophore upon titration with mild base has been developed. The maximum absorbance for the chromophore is at 260 nm with an extinction coefficient of 9700 m −1 cm−1. The method can be used for measuring the active esters on carriers and their interaction with amines during peptide synthesis.

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.

Thirty years of affinity chromatography

Affinity chromatography was introduced 30 years ago and is the most powerful tool for purification of biologically active molecules. Affinity chromatography has also had a major impact and virtually revolutionized the entire field of modern biology, molecular biology, biochemistry, medicine and biotechnology. The development of affinity chromatography has led to many other studies and applications in which the concept of molecular recognition and biorecognition is used.

On the mode of adsorption of proteins to “hydrophobic columns”

Abstract α-Lactalbumin and ovalbumin were found to bind tightly to alkyl amino agaroses with hydrocarbon chains of 4–6 carbon atoms. These proteins were not adsorbed when the columns were acetylated. The binding of protein to the hydrophobic column appears to occur in two phases. The first of these is suggested to involve binding of the protein anions to the cationic site on the columns. The second phase is suggested to involve binding of the hydrophobic portions of the column with the hydrophobic amino acid residues of the protein.

ALLICIN-INDUCED DECREASE IN FORMATION OF FATTY STREAKS (ATHEROSCLEROSIS) IN MICE FED A CHOLESTEROL-RICH DIET

BACKGROUND Garlic (Allium sativum) has been considered to exhibit therapeutic features for many years. The effects of garlic on levels of serum lipids and on atherosclerosis have been investigated extensively. We have previously demonstrated that allicin, an active component of garlic, exerts a beneficial effect on lipid profile in hyperlipidemic rabbits. OBJECTIVE To investigate the effects of allicin on formation of fatty streaks (atherosclerosis) and lipid profile in mice. METHODS Allicin was extracted from garlic and kept in a buffer citrate solution at 4 degrees C. Sixty C57BL/6 mice were fed Paigen diet (17% fat, 1.25% cholesterol) for 15 weeks. Thirty randomly selected animals were administered allicin solution (9 mg/kg) and 30 were administered placebo. Blood lipid profile was evaluated five times during the study. At the end of the 15-week period, the animals were killed and the aortic sinus was evaluated for formation of fatty streaks (atherosclerosis). RESULTS We observed no statistically significant differences between blood lipid profiles of groups. Microscopic evaluation of aortic sinus formation of fatty streaks (atherosclerosis), however, showed that values for mice in the allicin-treated group were significantly lower: areas of formation of fatty streaks (atherosclerosis) were 13,440 +/- 3310 and 23,410 +/- 3723 micron 2, respectively, for allicin-treated and control mice (means +/- SEM; P = 0.023). CONCLUSIONS These results indicate that allicin reduces formation of fatty streaks (atherosclerosis) in hyperlipidemic mice. These changes do not seem to occur through an alteration in blood lipid profile.

Oriented versus random protein immobilization

Immobilization of proteins on solid surfaces plays an important role in all the fields of modern biology. Two approaches are used in the immobilization of proteins: a random and an oriented mode of binding to solid matrices. In this note, we show that there is not much difference in using either mode of immobilization, since proteins usually bind to a matrix through only one or two bonds. This is demonstrated by the attachment of several proteins to CNBr-activated Sepharose through their lysines and the consequent conversion of those lysines to homoarginine upon treatment with ammonium hydroxide.