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PubMed1995

An equivalent site mechanism for Na+ and K+ binding to sodium pump and control of the conformational change reported by fluorescein 5'-isothiocyanate modification.

We have previously shown that two K+ must bind to cause the conformational change in Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase reported by fluorescein 5'-isothiocyanate modification and that the ratio of the macroscopic K+ dissociation constants is 4 [Smirnova, I. N., & Faller, L. D. (1993) J. Biol. Chem. 268, 16120-16123]. Since 4 is the ratio expected for random binding to two identical and independent sites, in this paper we propose a two-equivalent-site mechanism for Na+ as well as K+ binding to sodium pump and control of the conformational change between E1 and E2. The equivalent site mechanism is tested by fitting algebraic equations for the empirical first-order rate constant and for the magnitude of the fluorescence change observed in stopped-flow experiments to an array of data. The estimated rate constants for the conformational change at 15 degrees C are kf = 150 +/- 19 s-1 and kr = 0.13 +/- 0.03 s-1, and the estimated dissociation constants for K+ and Na+ from the E1 conformation of the enzyme are KK = 4.8 +/- 1.2 mM and KNa = 0.38 +/- 0.12 mM, in good agreement with estimates from published binding measurements. Although the possibility of ordered and anticooperative binding cannot be excluded, the equivalent site interpretation is supported by quasi-independent estimates of the macroscopic Na+ dissociation constants that give the ratio 4 predicted for binding to identical, noninteracting sites and by equivalent site interpretations of published transport experiments. Six alternative mechanisms are tested. Noncompetitive binding and mutually exclusive binding of the transported ions can be ruled out. Comparable fits to the data are obtained by three-equivalent-site mechanisms in which enzyme with either two or three K+ bound can undergo the conformational change. Therefore, this study quantitatively supports the conformational hypothesis by showing that a conformational change in sodium pump has the right properties to explain transport.

Animals↗
PubMed1993

Role of Mg2+ ions in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na+,K(+)-ATPase.

The role of Mg2+ in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na,K-ATPase has been studied by stopped-flow fluorometry. K+ causes a fluorescence quench that is reversed by Na+. The principal experimental observations are as follows: (1) Mg2+ decreases the apparent affinity of the enzyme for K+ but does not affect the maximum rate of the K+ quench. (2) The amplitude of the K+ quench depends hyperbolically on the K+ concentration, and the maximum amplitude is unaffected by the Mg2+ concentration. (3) The rate at which Na+ reverses the K+ quench depends inversely on the Mg2+ concentration. (4) The amplitude of the Na+ reversal also decreases with increasing Mg2+ concentration. The data are quantitatively explained by a model that assumes only two enzyme conformations, detectable by their fluorescence emission. Mg2+ increases Kd for K+ from 14 to 223 mM. At 22 degrees C, Kd = 0.16 mM for Mg2+ dissociation from E1, and the heat of Mg2+ binding, delta H degrees, is 11.4 kcal mol-1. Kd is more than an order of magnitude larger for Mg2+ dissociation from E2K. Mg2+ binding does not affect the forward (E1K-->E2K) rate constant (kf), but decreases the reverse rate constant (kr) thus increasing the equilibrium constant for the reaction (Kc = kf/kr) 6-fold. Therefore, Mg2+ is not directly involved in the conformational transition, but the study supports proposals that Mg2+ binding and release may help to regulate the transport cycle by shifting the distribution of enzyme between E1 and E2 conformers.

Animals↗
PubMed1991

Immunochemical and spectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on Na+,K(+)-ATPase.

Fluorescein 5'-isothiocyanate (FITC) covalently modifies the Lys-501 residue of the catalytic (alpha) subunit of Na+,K(+)-ATPase and resides at a conformation-sensitive site in or near the ATP binding site. In these studies, FITC-directed antibodies which quench this hapten's fluorescence were used to infer the solvent accessibility of the enzyme-bound probe. These antibodies identified two FITC labeling populations. An antibody-accessible population, representing 20-50% of the bound FITC fluorescence, was essentially (95%) quenched by the antibody. The second population was irreversibly labeled, was inaccessible to antibody, and was the fraction of probe whose fluorescence intensity is sensitive to the enzyme's conformation. The anti-FITC antibodies therefore permitted the selective investigation of FITC at this active site. Distinct differences between the two labeling sites were then demonstrated. Shifts in the absorption spectrum suggested that the active-site-bound probe resides in a hydrophobic environment, while polarization values indicated a rigid, rotationally restricted location. These two properties were not altered by ligand additions. Iodide quenching studies, however, showed that in the E1Na+ conformation there was a 50% decrease in solvent access to the active-site-bound probe as compared to free probe while the E1Na(+)----E2K+ transition decreased this accessibility an additional 50%. Similarly, there was a significant decrease in the relative quantum yield of FITC linked at this site that was reduced further by the E1Na(+)----E2K+ transition. In contrast, frequency domain spectroscopy showed no significant differences in the lifetimes of fluorescence decay for the two different labeling populations nor for the high (E1Na+) and low (E2K+) fluorescence intensity conformations. We have found that static (lifetime independent) quenching rather than collisional processes or protonation changes accounts for the fluorescence intensity changes undergone by FITC bound at the ATP-protectable site.

Animals↗
PubMed1984

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion.

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.

Animals↗
PubMed1985

Location of the maltosyl isothiocyanate binding site on the human erythrocyte glucose transporter.

The covalent affinity probe maltosyl isothiocyanate (MITC) has been used previously to identify the glucose transporter of human erythrocytes as a component of band 3. By use of limited proteolysis, the site on the Mr 100 000 protein to which MITC attaches has been localized to a 17 000-dalton region near the center of the polypeptide chain which is intimately associated with the membrane. The erythrocyte anion transporter, which is probably homologous to the glucose carrier, has a corresponding segment which is known to bind the covalent affinity label 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1980) Biochim. Biophys. Acta 559, 127-139]. These results suggest that, in addition to having structural features in common, the two carrier proteins may be quite similar with regard to functional organization.

Affinity Labels↗
PubMed1987

Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T alpha subunit. There was no significant incorporation on the T beta gamma subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp(NH)p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of T alpha and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp(NH)p complex was about 3-fold slower than that of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the T alpha subunit. These results indicate that FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of T alpha, which ultimately leads to the activation of cGMP phosphodiesterase.

Animals↗
PubMed1988

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate.

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.

Adenosine Monophosphate↗
PubMed1989

Interaction of calcium and vanadate with fluorescein isothiocyanate labeled Ca2+-ATPase from sarcoplasmic reticulum: kinetics and equilibria.

The interaction of Ca2+ and vanadate with fluorescein isothiocyanate (FITC) labeled sarcoplasmic reticulum (SR) Ca2+-ATPase has been studied by following the kinetics of changes in the reporter group fluorescence and equilibrium fluorescence levels. The vanadate species bound to the enzyme is clearly monomeric orthovanadate, probably H2VO4-. Vanadate binding is noncooperative, suggesting an absence of interactions between the Ca2+-ATPase subunits. The fluorescence experiments confirm the existence of a calcium-enzyme-vanadate complex (in the presence of magnesium). On the basis of the fluorescence properties of this complex, it is similar in its conformation to the calcium-enzyme complex, i.e., "E1-like" rather than "E2-like". However, Ca2+ binds to the enzyme-vanadate complex via sites that are only accessible from the interior of the SR vesicles. The complex Ca2E*Van, which is rapidly formed, isomerizes very slowly (t1/2 approximately 1 min) to the stable ternary complex. The mutual destabilization between bound vanadate and two bound Ca2+ ions is only 1.6 kcal/mol, much smaller than that produced by the interaction of calcium and phosphate.

Animals↗
PubMed1989

Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase.

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1980

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 1. Inhibition of glucose transport.

Maltosyl isothiocyanate (MITC) has been synthesized from maltose with an overall yield of 88%. It has been found to be a potent irreversible inhibitor of zero trans influx of glucose with human erythrocytes. Kinetic analysis of glucose transport after treatment of erythrocytes with MITC revealed that VT was diminished while KT was unchanged. Transportable sugars and competitive inhibitors of monosaccharide transport protected against MITC inhibition, while carbohydrates which do not interact with the transporter gave no protection. Covalent inhibitors of anion transport were without effect on glucose transport. MITC fulfilled the kinetic requirements for an affinity label of the glucose transporter of human erythrocytes [Groman, E. V., Schultz, R. M., & Engel, L. L. (1977) Methods Enzymol. 46, 54].

Affinity Labels↗
PubMed1980

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 2. Identification of the transporter.

Maltosyl isothiocyanate (MITC), a potent irreversible inhibitor of glucose transport in human erythrocytes [Mullins, R. E., & Langdon, R. G. (1980) Biochemistry (preceding paper in this issue)], has been found to react almost exclusively with band 3 of the human erythrocyte membrane. The incorporation of [14C]MITC into band 3 was found to be antagonized by transportable sugars or competitive inhibitors of transport. On the basis of [14C]MITC incorporation into band 3 and MITC inhibition of transport, it is estimated that there are 3 x 10(5) glucose transporters present in the erythrocyte membrane. It was found that [14C]MITC-labeled band 3 could be converted into 14C-labeled band 4.5 during the Triton X-100 extraction procedure described by Kasahara & Hinkle [Kasahara, M., & Hinkel, P. C. (1977) J. Biol. Chem. 252, 7384]. On the basis of the evidence presented here and in the preceding paper, it is suggested that in the native erythrocyte membrane a component of band 3 is the glucose transport protein and that during purification with nonionic detergents the transport protein may be enzymatically degraded with some retention of activity.

Affinity Labels↗
PubMed1980

Radiolabeling of natural adenosine triphosphatase inhibitor with phenyl (14C)isothiocyanate and study of its interaction with mitochondrial adenosine triphosphatase. Localization of inhibitor binding sites and stoichiometry of binding.

The natural ATPase inhibitor (IF1) from beef heart mitochondria was labeled with phenyl (14C)isothiocyanate [(14C)PITC]. Chemical labeling by (14C)PITC does not modify the inhibitory properties of IF1, provided the number of residues of (14C)PITC bound per molecule of IF1 is lower than five to six, which corresponds to the average labeling of roughly half of the available lysine residues in IF1. This partially labeled, fully active, IF1 was used to determine the binding stoichiometry of IF1 with respect to F1 and to localize the inhibitor binding sites in F1-ATPase. The pattern of loss of ATPase activity of F1 with increasing amounts ot (14C)PITC-IF1 indicated that the ATPase activity is fully inhibited when 1 mol of IF1 is bound to 1 mol of F1. As F1 contains at least 2 beta subunits, this points to a half-site reactivity of F1 with respect to IF1. Sites of interaction between (14C)PITC-IF1 and F1 subunits were investigated by the use of two cross-linking reagents which act as "zero length" cross-linkers, 1-ethyl-3-[(dimethylamino)propyl]carbodiimide (EDAC) and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline (EEDQ); the products of cross-linking were analyzed by NaDodSO4-polyacrylamide gel electrophoresis. IF1 was found to bind preferentially to the beta subunit of F1. Among the cross-linked products formed by reaction of EDAC or EEDQ with subunits of F1, one of them, the beta gamma dimer, did not accumulate when IF1 was added to F1 prior to cross-linking.

Adenosine Triphosphate↗
PubMed2001

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1.

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.

Animals↗
PubMed1998

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Adenosine Triphosphate↗
PubMed2006

Coaxial electrospinning of (fluorescein isothiocyanate-conjugated bovine serum albumin)-encapsulated poly(epsilon-caprolactone) nanofibers for sustained release.

As an aim toward developing biologically mimetic and functional nanofiber-based tissue engineering scaffolds, we demonstrated the encapsulation of a model protein, fluorescein isothiocyanate-conjugated bovine serum albumin (fitcBSA), along with a water-soluble polymer, poly(ethylene glycol) (PEG), within the biodegradable poly(epsilon-caprolactone) (PCL) nanofibers using a coaxial electrospinning technique. By variation of the inner flow rates from 0.2 to 0.6 mL/h with a constant outer flow rate of 1.8 mL/h, fitcBSA loadings of 0.85-2.17 mg/g of nanofibrous membranes were prepared. Variation of flow rates also resulted in increases of fiber sizes from ca. 270 nm to 380 nm. The encapsulation of fitcBSA/PEG within PCL was subsequently characterized by laser confocal scanning microscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) analysis. In vitro release studies were conducted to evaluate sustained release potential of the core-sheath-structured composite nanofiber PCL-r-fitcBSA/PEG. As a negative control, composite nanofiber PCL/fitcBSA/PEG blend was prepared from a normal electrospinning method. It was found that core-sheath nanofibers PCL-r-fitcBSA/PEG pronouncedly alleviated the initial burst release for higher protein loading and gave better sustainability compared to that of PCL/fitcBSA/PEG nanofibers. The present study would provide a basis for further design and optimization of processing conditions to control the nanostructure of core-sheath composite nanofibers and ultimately achieve desired release kinetics of bioactive proteins (e.g., growth factors) for practical tissue engineering applications.

Animals↗
PubMedDate not supplied

Trityl isothiocyanate support for solid-phase synthesis.

Trityl isothiocyanate resin [1], prepared from commercially available trityl chloride resin, is a useful precursor of the trityl thiosemicarbazide resin [2]. This resin can be employed in the solid-phase synthesis of a variety of supported isatin beta-thiosemicarbazones [4] and their Mannich derivatives [6]. A variety of thioureas [7] can be easily prepared by the reaction of [1] with amines. The supported thioureas are directly and efficiently converted to 2-aminothiazole-5-carboxylates [8] by reaction with methyl 2-chloroacetoacetate.

Combinatorial Chemistry Techniques↗
PubMed2001

Glucosinolate content and isothiocyanate evolution--two measures of the biofumigation potential of plants.

A total of 570 lyophilised Brassica root and shoot tissue samples were hydrolyzed, and the liberated isothiocyanates (ITCs) were analyzed by gas chromatography-flame photometric detection (GC-FPD). Glucosinolates (GSLs) were extracted from samples of the same tissues and analyzed by high-performance liquid chromatography (HPLC). The concentrations of six GSLs/ITCs (2-propenyl, 3-butyl, 4-pentenyl, benzyl, 4-methylthiobutyl, and 2-phenylethyl) as determined by the two techniques were compared. In 79% of the samples, the concentration of GSLs in the tissues was greater than that of the ITCs released on hydrolysis. Several possible reasons for the difference are proposed, including the effect of tissue storage time, hydrolysis of GSLs may be less efficient than the GSL extraction procedure, or some of the ITCs formed reacted with plant proteins and amino acids in the sample and were therefore not detected in the extract. GSL concentration in plant tissues is used to estimate the biofumigation potential of the plant tissue, whereas the actual biofumigation effect is thought to be due to the ITCs formed by hydrolysis of the plant-based GSLs. The variation between ITC and GSL values therefore has implications for the assessment of the biofumigation potential of the plant tissue.

Brassica↗
PubMed2002

Direct and simultaneous analysis of sinigrin and allyl isothiocyanate in mustard samples by high-performance liquid chromatography.

A reversed-phase HPLC method for the simultaneous determination of the glucosinolate sinigrin and its major degradation product allyl isothiocyanate (AITC) was developed and used for direct analysis of aqueous extracts from Oriental mustard (Brassica juncea L.) related materials (ground and cracked seeds, powders, and bran) and from soil samples. The lowest detection limit was 0.1 microg/mL for both sinigrin and AITC). The developed method was used to trace the degradation of sinigrin to AITC in aqueous extracts. One of the major advantages of this method is the complete estimation of sinigrin content. The simultaneous analysis of both sinigrin and AITC in a single run avoided the underestimation caused by separate analyses.

Acetates↗