Low Buffer Concentrations Research Articles

Polyfunctional ( [formula omitted])-2-hydroxycarboxylic acids by reduction of 2-oxo acids with hydrogen gas or formate and resting cells of proteus vulgaris

Various ( R )-2-hydroxy acids such as ( R )-2-hydroxy-3-enoic-, 3,5-dienoic-, 4-oxo-, ( R,S )-3-hydroxy and some others were prepared on a scale up to 0.12 mol by biocatalytic reduction of the corresponding 2-oxo acids with P. vulgaris and hydrogen gas and/or formate as electron donors. With the exception of the 2-hydroxy-4-oxo acids it could be proved that the enantiomeric excess is > 97%. For the 4-oxo derivatives this enantiomeric excess can be assumed. The yields of isolated products are high because they were isolated from rather small amounts of biocatalyst and low buffer concentrations, product concentrations in the range of 0.1–0.24 M were obtained. For 1 mmol of product formation in 15–20 h about 20–40 mg (dry weight) of P. vulgaris cells are necessary.

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides: Instrumental considerations for mass spectrometric detection

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-μm I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection, and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15–20 m M) of ammonium acetate or ammonium formate, and high organic modifier content (5–50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0–0.04 μl/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous ‘infusion’ of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.

Kinetics of reaction of 1,2-diaminobenzene with phenylglyoxal

The cyclization reaction kinetics of phenylglyoxal monohydrate with 1,2-diaminobenzene have been studied in formate, acetate, and phosphate buffers. At high pH values and low buffer concentrations the rate-limiting step consists in the protonation of the intermediate formed by addition of the first amino group to aldehydic group of phenylglyoxal. With increasing concentrations of formate and acetate buffers the rate-limiting step shifts to the formation of the intermediate. In phosphate buffers the catalysis by the basic buffer component makes itself felt, too. At higher concentrations of 1,2-diaminobenzene, the dehydration of phenylglyoxal monohydrate gradually becomes the rate-limiting step.

Isolation and Properties of Rat Plasma Lecithin-Cholesterol Acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.

ChemInform Abstract: Kinetic Evidence for the Occurrence of a Stepwise Mechanism in the Aminolysis of N‐Ethoxycarbonylphthalimide.

AbstractThe aminolysis of (I) with (IIa) in the presence of acetate shows a sharp (slow) increase in the reaction rate at low (considerably higher) total buffer concentrations, due to a change in the rate‐determining step.

The mechanism of the nonenzymatic iodination of tyrosine by molecular iodine

Over the pH range 7–10, at very low buffer concentration, the nonenzymatic iodination of tyrosine obeys the rate law[Formula: see text]where kapp is the measured second order rate constant based upon the total initial concentrations of molecular iodine and tyrosine and K2 (units M) is the equilibrium constant for [Formula: see text]. The value of k′ is 3.5 × 10−8 M∙s−1. There are three plausible mechanisms that fit the experimental data. One, the simplest, is a concerted process in which hypoiodous acid attacks tyrosine with its phenolic group unionized. The other two involve the formation of an iodinated quinoid reactive intermediate species in a rapid pre-equilibrium between unionized tyrosine and either hypoiodous acid or molecular iodine. The pre-equilibrium, if it occurs, favors the initial reactants. It is followed by a slow step in which the quinoid is converted to mono-iodinated tyrosine. Positive deviations from the rate law for pH dependence indicate that some specific acid catalysis (H3O+) is occurring in the pH range 5–7. In the presence of sufficient buffer, general acid–base catalysis is observed with acetic acid acting as a general acid catalyst in the vicinity of pH 5 and carbonate acting as a general base at pH ~ 9.5. The nonenzymatic iodination of tyrosine occurs more rapidly as the pH is increased, in marked contrast to the peroxidase-catalyzed iodination, which has its optimum at low pH.

Origin of viscosity effects in carbonic anhydrase catalysis. Kinetic studies with bulky buffers at limiting concentrations.

In our earlier paper we showed that the rates of CO2 hydration and HCO3- dehydration catalyzed by the high-activity form of mammalian erythrocyte carbonic anhydrase (CA II) were dependent on solution viscosity increase and that the effect was linked to some kind of proton-transfer-related event [Pocker, Y., & Janjić, N. (1987) Biochemistry 26, 2597-2606]. In order to further elucidate the source of the observed viscosity effect, the dependence of kcat and Km for CA II catalyzed HCO3- dehydration at pH 5.90 on sucrose-induced viscosity increase was investigated at several concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, including the very low buffer concentration region (less than 10 mM) where the proton transfer between the shuttle group on the enzyme and buffer becomes rate limiting. In all examined cases, kcat steadily decreased with added sucrose while Km remained independent of the viscosity increase. The extent to which this reaction was dependent on viscosity was found to be constant, within experimental error, over the entire range of MES buffer concentrations studied (1-20 mM). Furthermore, the viscosity effect was qualitatively and quantitatively the same when an exceptionally large buffer (i.e., bovine serum albumin) was used instead of the more commonly used biological buffer (i.e., MES).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum.

1. Mast cells, isolated from rat peritoneum, were studied under tight-seal, whole-cell recording conditions. Membrane conductance, membrane capacitance and the concentration of free intracellular Ca2+, [Ca2+]i, were measured simultaneously. 2. [Ca2+]i could be accurately buffered to values between 0 and 1.5 microM only if relatively high concentrations of calcium buffers (in the millimolar range) were added to the pipette filling solution against which the cytoplasm was dialysed. At lower buffer concentrations [Ca2+]i was markedly increased by hyperpolarizing the membrane. 3. When added to the pipette, guanosine-3-thio-triphosphate (GTP-gamma-S), a nonhydrolysable analogue of guanosine triphosphate, stimulated a 3.3-fold increase in membrane capacitance, which is indicative of mast cell degranulation (Fernandez, Neher & Gomperts, 1984). 4. In weakly buffered cells, GTP-gamma-S also induced a transient increase in [Ca2+]i which, usually, preceded degranulation. Calcium buffers at 1-5 mM concentration suppressed this transient. 5. High [Ca2+]i alone did not induce degranulation. However, it markedly accelerated GTP-gamma-S-induced degranulation. When [Ca2+]i was buffered to zero, an appreciable fraction of cells degranulated in response to GTP-gamma-S, but very slowly, and only after a long lag phase. 6. Transient increases in [Ca2+]i, evoked either by GTP-gamma-S, or by voltage changes, did not elicit capacitance changes during the lag phase, but accelerated the GTP-gamma-S-induced degranulation response at later times. 7. Internally applied inositol 1,4,5-trisphosphate (IP3) also induced transient increases in [Ca2+]i which did not lead to secretion in the absence of GTP-gamma-S. 8. It is concluded that an increase in [Ca2+]i is neither necessary nor sufficient for secretion from dialysed mast cells. [Ca2+]i, however, acts synergistically with other stimuli to promote secretion. It is the more efficient the more time the other stimulus had been allowed for priming the cell.

Substituent effects and isotope effects on the rate coefficients and equilibrium constant for opening an intramolecular hydrogen bond

Proton removal from the intramolecularly hydrogen-bonded monoanions of substituted 4,6-bis(phenylazo)resorcinols in buffered 70%(v/v) Me2SO–H2O involves opening of the hydrogen bond followed by proton transfer between open form and buffer. At low buffer concentration the proton transfer is rate-limiting but opening of the hydrogen bond becomes rate-limiting at higher buffer concentration. Typical values of the rate coefficients for opening (k1) and closing (k–1) of the hydrogen bond are k1 3.8 ± 0.6 × 104 s–1 and k–1ca. 8 × 107 s–1 found for 4,6-bis(phenylazo)resorcinol monoanion. The rate coefficients and the corresponding equilibrium constant are almost unaffected by substituents and by changing the solvent to 70%(v/v) Me2SO–D2O.

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA.

AbstractThe results of semiselective 1H‐nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D2O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A‐T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s−1 at 25°C. Corresponding values for the G‐C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A‐T and G‐C imino protons, resembling the effect recently observed for poly(rA)‐poly(rU) and poly(rI)‐poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base‐pair opening rates as assumed in the past. Lower limits to the base‐pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 103 s−1 for the A‐T, and 50 s−1 for the G‐C, base paris at 62°C.

Multispecific anion exchange in basolateral (sinusoidal) rat liver plasma membrane vesicles.

The mechanisms and driving forces for hepatic uptake of sulfate were investigated in basolateral (sinusoidal) rat liver plasma membrane vesicles. A transmembrane pH difference (pH 8.0 inside, 6.0 outside) stimulated sulfate uptake above equilibrium ("overshoot"). This pH gradient-stimulated sulfate uptake was saturable with increasing concentrations of sulfate and could be inhibited by probenecid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone, and nigericin. At low buffer concentrations and pH 6.0 an inwardly directed sodium gradient also stimulated sulfate uptake. This sodium-dependent sulfate uptake could be inhibited by amiloride and DIDS, indicating indirect coupling of sodium and sulfate flux through concomitant sodium-proton and sulfate-hydroxyl exchange. Cisinhibition of initial pH gradient-stimulated sulfate uptake, as well as transstimulation of sulfate uptake under pH-equilibrated conditions (pH 7.5 inside and outside), were observed with sulfate, thiosulfate, oxalate, and succinate, but not with chloride, bicarbonate, acetate, lactate, pyruvate, p-aminohippurate, citrate, glutamate, aspartate, and taurocholate. Furthermore, cholate and sulfobromophthalein exhibited competitive inhibition of pH gradient-stimulated sulfate uptake. In addition, an inside-to-outside hydroxyl gradient also stimulated uptake of cholate and this pH gradient-sensitive portion of cholate uptake was inhibited by extravesicular sulfate. In contrast to basolateral membranes, no evidence for multispecific sulfate-hydroxyl exchange was found in canalicular plasma membrane vesicles.

On the product distribution in the iodination of phenol

The product distribution in the iodination of phenol with I 2, in water solution is buffer and pH dependent with the ortho product increasing at low pH and low buffer concentration. This effect is attributed to different rates of proton transfer from the 4-iodo-2,5-; and 2-iodo-3,5-cyclohexadienone intermediate.

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 m) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H 2O and D 2O were used as solvent. For both the addition and the elimination of SO 3 2− values of k 2 H k 2 D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 m, pH 7.2, are 10.2 and 1.3 m −1, respectively.

Continuous assay method for glycogen synthase activity

An assay method for glycogen synthase (EC 2.4.1.11) has been developed based on the continuous measurement of the change of pH accompanying the glycogen synthesis reaction. The use of low buffer concentrations and an amplifier with variable gain and offset voltage allow us to register changes in the pH of the system small enough to ignore the significant pH dependence of the enzyme activity. A theoretical approach has been used to correlate the pH measurements with the progres of the reaction in terms of glucose incorporated into glycogen. The method offers the advantages of being continuous and of low cost.

Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling.

An inside-alkaline pH gradient (pH 7.7 inside, 5.5 outside) stimulated Cl uptake in brush-border vesicles from rabbit ileum. The addition of HCO3 without changing the pH gradient further stimulated Cl uptake to a level fourfold greater than equilibrated Cl uptake. Although a K diffusion potential stimulated Cl uptake, this was insensitive to inhibition by 4,4-diisothiocyanostilbene-2,2'-disulfonate (DIDS), whereas pH and HCO3 gradient-stimulated Cl uptake was inhibited by DIDS. pH and HCO3 gradient-stimulated Cl uptake was found to be a saturable function of the Cl concentration with a Km of 3.5 mM and a Vmax of 49 nmol X mg prot-1 X min-1. To distinguish between coupling of Na and Cl transport by cotransport or dual exchange (Na-H and Cl-HCO3 exchange), we determined uptake with high (134 mM Tris-HEPES-MES) internal buffer and low (1.34 mM Tris-HEPES-MES) internal buffer concentrations. Inwardly directed gradients of 50 mM Na, 50 mM K, or 50 mM Na and K did not stimulate Cl uptake, and 50 mM Cl, 50 mM K, or 50 mM KCl did not stimulate Na uptake, with high internal buffer, excluding cotransport. In contrast, 50 mM Na stimulated Cl uptake (inhibited by 1 mM DIDS) and 50 mM Cl stimulated Na uptake (inhibited by 1 mM amiloride) in low buffer media. To determine a role for carbonic anhydrase, Na-stimulated Cl uptake was determined in low buffer media, equilibrated with either 100% N2 or 95% N2-5% CO2. Na stimulated Cl uptake 80% (compared with trimethylammonium control) with CO2 but only 30% with N2 (P less than 0.05). Acetazolamide partially inhibited (P less than 0.025) the stimulation of Cl uptake with CO2 but not with N2. Carbonic anhydrase activity was measured in homogenate and brush-border membrane and was enriched 7.9 +/- 0.4-fold, whereas sucrase was enriched 14.0 +/- 1.1-fold. We conclude that coupled Na and Cl transport occurs by dual exchange (Na-H and Cl-HCO3) and carbonic anhydrase, apparently located on the brush-border membrane, facilitates dual exchange by providing HCO3.

Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase.

delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione.

Effect of buffers on the functional properties of Helix pomatia β-hemocyanin

The possibility that chemicals commonly used as buffers may affect the functional properties of Helix pomatia β-hemocyanin has been investigated. Bistris-HCl, Tris-HCl and Na-Hepes change the shape and position of the oxygen-binding curves in different ways. Bistris and Tris cations increase the oxygen affinity, while Hepes anions show the opposite effect. The effect of Tris cations is only evident at low buffer concentrations (below 50 mM), since at higher buffer concentration the opposite effect of chloride becomes dominant. Sodium ions affect the binding properties to a minor extent. Analysis of these data within the framework of the Monod-Wyman-Changeux model indicates that Tris cations bind preferentially to the high oxygen-affinity (R)-state of the protein, while additional effects on the T-state are observed. Hepes and chloride anions, on the other hand, show the opposite behavior, interacting preferentially with the T-state. Thus specific effects of buffer components on the oxygen-binding equilibrium of hemocyanins have to be taken into account in a quantitative analysis of linkage phenomena in these extended systems. This type of phenomenon may be more common than previously anticipated.

The catalytic mechanism of carbonic anhydrase

Steady-state and equilibrium kinetic studies of the CO 2HCO − 3 interconversion catalyzed by human carbonic anhydrase II (or C) have led to the proposal of a mechanism scheme (Scheme 1) involving two ionizing groups [1–4]. One of these is probably a zinc-bound H 2O molecule ionizing to an OH − ion which can react with CO 2 to form zinc-bound HCO − 3. The other group is probably His-64 which is located in the hydrophilic part of the active site at some distance from the zinc ion. At low buffer concentrations the rate-limiting step in catalysis is the transfer of H + between His-64 and buffer molecules. At high buffer concentrations the rate-limiting step seems to be an intramolecular H + transfer between the two active-site groups: HisEZn 2+OH 2 ⇌ +HHisEZn 2+OH −. When Scheme 1 was first proposed [1] it was assumed that the pK a values of the two ionizing groups were identical and that there was no interaction between the groups. However, it has later been shown that the early results were affected by SO 2− 4 ions which inhibit at low pH [5]. When the effects of SO 2− 4 are taken into account it must be assumed that there is, indeed, an interaction between the two active-site groups so that they do not operate independently of one another. The rate equation describing the steady-state velocity resulting from Scheme 1 is exceedingly complex. Rather than use such an equation, we have computer simulated the kinetic behaviour predicted by Scheme 1 using various sets of rate constants. It will be shown by this method that Scheme 1 can describe satisfactorily the known kinetic behaviour of human carbonic anhydrase II. It will also be shown that Scheme 1 predicts the deviations from Michaelis-Menten kinetics observed for the low-activity human carbonic anhydrase I (or B) under certain conditions.

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase.

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.

Adsorption of some .BETA.-glycosidases on N-(.EPSILON.-aminohexanoyl)-.BETA.-D-glycosylamine-agarose.

Adsorption of β-galactosidase (EC 3. 2. 1. 23), β-glucosidase (EC 3. 2. 1. 21) and β-acetyl-glucosaminidase (EC 3. 2. 1. 30) on agarose substituted with N-(e-aminohexanoyl)-β-D-galactosylamine, -β-D-glucosylamine, -2-acetamido-2-deoxy-β-D-glucosylamine and -β-D-mannosylamine was studied. All the glycosidases were adsorbed only at low buffer concentration. Although the adsorptions of Taka-diastase and Sanactase glycosidases were non-biospecific, the adsorption required the presence of the glycone as a ligand and the elution patterns in column chromatography were affected by variation of the glycone moiety of the adsorbent. The adsorptions of the enzymes on galactosylamine-agarose were prevented by low concentrations of hexoses and pentoses. The adsorption and desorption of emulsin glycosidase were apparently biospecific considering that the enzyme protein has one binding site for glucoside and one for galactoside, although the biospecificity was inexplicable in view of the large K1 values of the spacerligand compounds.