Essential Thiol Groups Research Articles

Mitochondrial phosphate carrier. Functional role of its SH groups and interrelations within the carrier unit.

1. The phosphate carrier of isolated rat liver mitochondria was studied. 2. To investigate the effect of the substrate on the external side of the transport protein, a technique was required by which the extramitochondrial phosphate concentration could be changed without any effect on either the phosphate content or the pH value in the intramitochondrial compartment. This was found when non-respiring mitochondria were incubated with the ionophore nigericin. 3. Using this method variation of the phosphate concentration outside the mitochondria did not protect the essential SH groups located at the external surface of the phosphate carrier. 4. Interrelations within the phosphate carrier unit were investigated in mitochondria in which only one of the two essential thiol groups of the carrier unit was free. These mitochondria were however able to carry out phosphate transport. 5. Alterations of the intramitochondrial pH-induced modification of the position of the single free sulfhydryl group at the external surface of the membrane similarly to the changes which occur in intact mitochondria where both SH groups are free [E. Ligeti and A. Fonyó (1984) Eur. J. Biochem. 139, 279-285]. 6. It is suggested that the sulfhydryl groups essential for the transport function do not participate in the formation of the substrate binding site of the carrier protein. They probably have a role in the conformational change necessary for the translocation step. The possibility of two equivalent but independently functioning parts within the carrier unit is raised.

Synthesis of Enkephalin Analog with Leucinthiol at C-Terminus as Probe for Thiol Group in Opiate Receptors

Abstract Enkephalin analog with thiol group at C-terminus, [D-Ala2, Leu(CH2SH)5]enkephalin, was synthesized as possible probe for the essential thiol group in opiate receptors. In order to protect the free SH group of leucinthiol, oxidized Boc-l-leucinthiol dimer was prepared from l-leucinol (2-amino-4-methyl-1-pentanol) to elongate the enkephalin sequence. SH-Containing monomeric enkephalin analog was obtained from dimer by treatment with zinc in 50% AcOH, and it exhibited a high affinity for mu opiate receptors.

Metabolism of glycerate 2,3-P 2—XI. Essential amino acids of pig phosphoglycerate mutase isozymes

Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. 1. 2. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. 2. 3. The reversion of the modifying reactions reactivates the three enzymatic activities. 3. 4. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. 4. 5. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. 5. 6. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. 6. 7. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.

Thiol-containing enkephalins for exploration of the essential thiol group in opiate receptors.

Thiol-containing enkephalins for exploration of the essential thiol group in opiate receptors.

Disulfide bond formation between the active-site thiol and one of the several free thiol groups of chymopapain.

Chymopapain, a cysteine protease of papaya latex, has been purified with the use of fast protein liquid chromatography. Two homogeneous fractions were analyzed for thiol content and thiol reactivity. It was found that peak 1 and peak 2 contained two and three thiol groups, respectively, per mole of enzyme. This result is inconsistent with the general belief that chymopapain contains one essential and one nonessential thiol group and suggests that a significant portion of the thiol groups was oxidized in the previous preparations. Such an oxidation can account for some of the inconsistent results reported in the literature. An irreversibly oxidized nonessential thiol group may modify the catalytic function of chymopapain especially if it is close to the active site. That one thiol group resides indeed in the vicinity of the essential thiol group is clearly demonstrated by the biphasic reactions of chymopapain with disulfide compounds such as 2,2'-dipyridyl disulfide and 5,5'-dithiobis(2-nitrobenzoate). In the first step of these reactions a mixed disulfide is formed between the enzyme and the reactant, which is followed by a first-order, intramolecular reaction leading to the liberation of the second half of the disulfide compound. Furthermore, on addition of one Hg2+ ion, 2 mol of thiol group, one essential and one nonessential, disappears concomitantly. Formation of a disulfide bond between the catalytically competent thiol group and another free thiol group of chymopapain under physiological conditions may be of regulatory importance.

Identification of the essential cysteine residue of NADH-cytochrome b5 reductase.

The soluble catalytic domain of NADH-cytochrome b5 reductase was radiolabeled with [14C]N-ethylmaleimide. Reaction for a limited time resulted in incorporation of 0.41 eq of N-ethylmaleimide and loss of 36% of the enzyme activity. Chromatography on a 5'-ADP affinity column separated the reductase which was modified with N-ethylmaleimide from the unreacted enzyme; the isolated derivative constituted 37% of the total material, was completely inactivated, and contained 1.00 eq of N-ethylmaleimide. Cyanogen bromide cleavage of the derivative demonstrated that radioactivity was limited to a single peptide which contained both Cys-283 and Cys-297. Tryptic hydrolysis of this cyanogen bromide peptide showed that the radioactivity was associated with Cys-283. Automated sequenator analysis confirmed that Cys-283 was the radiolabeled residue. These data demonstrate unambiguously that Cys-283 provides the essential thiol group of cytochrome b5 reductase.

The time-dependent inactivation of human brain dihydropteridine reductase by the oxidation products of L-dopa.

Dihydropteridine reductase (DHPR) was irreversibly inactivated in a time-dependent way by incubation with 3,4-dihydroxyphenylalanine (L-dopa). The inactivation was oxygen-dependent; incubation under nitrogen gave partial protection. The inactivation was stimulated by the presence of horse-radish peroxidase/hydrogen peroxide. L-Dopa itself was not an inhibitor of DHPR although dopachrome, the aminochrome formed following oxidation of L-dopa, was a reversible inhibitor of DHPR with an I50 of 0.60 mM. The quinone products of oxidation of L-dopa were responsible for the time-dependent inactivation of DHPR. Adrenochrome also demonstrated a time-dependent inactivation of DHPR. Inactivation by adrenochrome demonstrated a saturation effect suggesting the reversible formation of a complex preceding inactivation. No radiolabel was incorporated into DHPR following inactivation by L-[14C]-dopa. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated the presence of a dimer of DHPR. A mechanism of inactivation involving the oxidative coupling of essential thiol groups was proposed to explain inactivation.

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

Modulation of activity of human alkaline phosphatases by Mg 2+ and thiol compounds

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg 2+ were studied. l-Cysteine (0.1 mmol/l) or Mg 2+ activated the liver enzyme 4–5-fold and the placental enzyme 2–3-fold, with optimal pH 7.5–8.0; these activations were not additive. l-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH > 9.0 ; phosphate protected the enzymes. S- Methylcysteine had little effect, with or without Mg 2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn 2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p- chloromercuribenzoate had little effect. The inhibition was reversed by l-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with l-cysteine; a Mg 2+-binding site, which results in activation, and a site that involves one or both of the bound Zn 2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.

Interactions between selenium and tin, selenium and lead, and their effects on alad activity in blood

Interactions of tin and selenium, as well as of lead and selenium, were investigated in male ICR mice. The toxic effects of selenium on mortality and body weight loss were reduced by simultaneous injection with tin or lead; among mice that were injected ip with selenium at 100 μmol/kg, the 24 h survival rate was 20%, whereas among mice that were administered selenium and tin or selenium and lead at the dose of 100 μmol/kg each, the rates were 100% and 90%, respectively. As for δ-aminolevulinic acid dehydratase (ALAD, EC 4.2.1.24), lead and tin were strong inhibitors, which is well known; selenium showed no effect. When more than an equimolar dose of sodium selenite was injected ip simultaneously with stannous chloride, the ALAD activity was completely retained. On the other hand, in the simultaneous injection with sodium selenite and lead acetate of differing ratios (Se/Pb), 1, 2.5, 5, and 7.5, selenium did not exhibit an obvious protective effect against the inhibition of ALAD activity caused by lead. It is suggested that selenium protects essential thiol groups in ALAD that are otherwise blocked by invading tin; in contrast, selenium, under similar conditions, does not prevent interactions of lead with enzyme thiol groups.

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.

Extended-X-ray-absorption-fine-structure investigations of zinc in 5-aminolaevulinate dehydratase.

The zinc co-ordination in 5-aminolaevulinate dehydratase (5-aminolaevulinate hydro-lyase, EC 4.2.1.24) was investigated by recording and interpreting the extended X-ray-absorption fine structure (e.x.a.f.s.) associated with the zinc K-edge. The enzyme has a molecular mass of 280 000 Da and consists of eight subunits of 35 000 Da each; the samples studied contained approx. 1 g-atom of zinc/mol of subunit. Four forms of the enzyme were investigated and details of the zinc environment were elucidated, as follows. In the native enzyme, zinc is considered to be co-ordinated to three sulphur atoms at 0.228(2)nm [2.28(2)A] and a lower-Z atom at 0.192(5)nm [1.92(5)A] (if nitrogen) or 0.189(5)nm [1.89(5)A] (if oxygen). Reaction of the enzyme with the inhibitor 2-bromo-3-(imidazol-5-yl)propionic acid produced significant changes in the e.x.a.f.s., the nature of which are consistent with co-ordination by about three sulphur atoms at 0.222(2)nm [2.22(2)A], a nitrogen atom at 0.193(5)nm [1.93(5)A] and a nitrogen atom from the inhibitor at 0.214(5)nm [2.14(5)A]. Inactivation of the enzyme by air-oxidation of essential thiol groups and binding of the substrate produce slight changes in the e.x.a.f.s. consistent with slight re-arrangement of ligands with additional lighter ligands (nitrogen or oxygen). These results, when combined with previous findings, are taken to indicate that zinc has a structural rather than a direct catalytic role in 5-aminolaevulinate dehydratase.

Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.

The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark‐grown seedlings of barley (Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5‐aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5‐aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5‐aminolaevulinic acid.

Functional and Regulatory Properties of H+ Pumps at the Tonoplast and Plasma Membranes of Zea mays Coleoptiles

Abstract A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of different membrane species. The membrane fractions were characterized by assaying commonly used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent H+ transport, and co- and countertransport of ions, such as Cl- , fumarate2-, K+ and Li+. The following results were obtained: (1) ATP hydrolysis is performed by different enzymes associated with different membranes: - vacuolar acid phosphatase (AP; inhibited by molybdate); - Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by molybdate); - ATPase activity of residual submitochondrial particles (sensitive to azide); - a H+-translocating ATPase at tonoplast membranes (Km(ATP) = 0.29 mᴍ; pH 7.5; stimulated by uncouplers and completely inhibited by NO- 3); - the H+-translocating ATPase of the plasmalemma (Km(ATP) = 0.39 mᴍ at pH 6.5, inhibited by vanadate, but not by NO- 3). The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily exist as inside-in-vesicles. (2) ATP-fueled H+ pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles, they differ to some extent in their properties: (a) The PLH+ pump has a very narrow pH optimum and exhibits highest levels of activity at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties, obviously important in vivo for the regulation of active H+-extrusion by certain growth substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press); in contrast, TO H+ pumps show a considerably wider pH optimum with highest levels of activity around pH 7.5. (b) In variance to the PL H+ pump the activity of TO pumps (Km(ATP) = 0.24 mᴍ) is regulated via the oxidation state of essential thiol groups. Their oxidation to the S -S - form (e.g. by blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH or cystein restores the activity [51]. (c) The ATP-fueled H+ transport into TO vesicles depends on an anion co-transport; most effective is Cl- , but there is also a stimulation by organic ions, C4 and C5 dicarboxylates, such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO- 3 is inhibitory. (d) H+-transport into sealed PL vesicles is also anion dependent. In this case, however, NO- 3 is as effective as Cl-. (3) The TO membranes contain a H+/K+ exchange mechanism responsible for a secondary active K+ uptake into the vacuole. This mechanism could be the reason for a lower (ATP dependent) acidification of TO vesicles in the presence of K+ compared with Li+. - Similar effects are observed with plasmamembrane vesicles, but in this case there is still the question whether a H+/K+-exchange, a K+ channel, or both are acting.

Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos

Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-(14)C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2- 14C]benzamido-2-deoxy-α- d-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with K assoc = 1.44 ± 0.13 × 10 3 m −1 (T = 5 °C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.

Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii.

Glyoxylate dehydrogenase (glyoxylate:NAD+ oxidoreductase) was purified 600-fold in three steps from crude extracts of the fungus Sclerotium rolfsii (Corticium rolfsii Curzi). Two of the purification steps involved dye-affinity chromatography. The enzyme is a tetramer of Mr 250 000, with identical subunits of Mr 57 000. Inhibition studies suggest that there is one essential thiol group per active site.

Effect of carbohydrate and metal ion binding on the reactivity of the essential thiol groups of lima bean lectin.

A free sulfhydryl group previously has been shown to be required for carbohydrate binding to the lectin from lima bean (Phaseolus lunatus) (Gould, N. R. and Scheinberg, S. L. (1970) Arch. Biochem. Biophys. 141, 607-613). Modification of this group by sulfhydryl reagents was specifically inhibited by D-GalNAc. We have further examined the reactivity of sulfhydryl groups in lima bean lectin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) as a probe for carbohydrate and metal ion binding. The 4 thiol groups in tetrameric lima bean lectin component III gave identical kinetics for reaction with Nbs2 involving formation of a weak noncovalent complex between Nbs2 and the lectin. The pH-independent reactivity of the thiol groups at neutral pH suggested that the thiols may exist as ion pairs with a nearby ionized group. Carbohydrate ligands were competitive inhibitors of thiol modification. The thiol groups on all 4 subunits of lima bean lectin were completely and reversibly protected by carbohydrate binding. The ability of carbohydrates to inhibit thiol modification correlated with their potency as inhibitors in a precipitin inhibition assay. The best inhibitors were the oligosaccharides alpha-D-GalNAc-(1 leads to 3)[alpha-L-fucose-(1 leads to 2)]beta-D-Gal(1 leads to R) and alpha-D-GalNAc-(1 leads to 2)beta-D-Gal(1 leads to R). Apparent thermodynamic parameters for binding of several carbohydrates were determined by measuring the temperature dependence of thiol protection. Removal of the bound metal ions Ca2+ and Mn2+ following dialysis into EDTA inactivated the lectin and increased the reactivity of the thiol groups 60-fold. This conversion was temperature-dependent and could be reversed upon addition of metal ions. The fast-reacting thiol groups were not protected by haptenic sugars from modifications by Nbs2.