Purification and characterization of a latent polyphenol oxidase from beet root (Beta vulgaris L.)

Polyphenol oxidase (PPO) has been extracted from beet root, in both soluble and membrane fractions. In both cases, the enzyme was in its latent state, and it was activated by sodium dodecyl sulfate. PPO was purified to apparent homogeneity. The soluble PPO purification was achieved by hydrophobic interaction chromatography and gel filtration chromatography, with apparent molecular mass of 55 kDa. The membrane PPO purification was achieved by anion exchange chromatography and gel filtration with apparent molecular mass of 54 kDa. A totally denaturing SDS-PAGE indicated the presence of a single polypeptide with an apparent molecular mass of 60 kDa for both fractions, with the band also revealed by Western blot. A partially denaturing SDS-PAGE stained a single active 36 kDa band for both fractions. Under native isoelectric focusing, a major acidic band of pH 5.2 was detected in both fractions. Kinetic characterization of PPO on the natural substrate l-dopa was carried out.     

Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase: purification and characterization

Polyphenol oxidase from iceberg lettuce (Lactuca sativa L.) chloroplasts was released from the thylakoid-membrane by sonication, and it was extensively purified to homogeneity as judged by SDS-PAGE. Purification was achieved by ammonium sulfate fractionation, gel-filtration chromatography, and ion-exchange chromatography. Two molecular forms were separated by gel-filtration chromatography with apparent molecular masses of 188 and 49 kDa. Both forms were characterized by sedimentation analysis with S(20,W) values of 10.2 and 4.1 S, respectively. For the high-molecular-weight form purified to homogeneity, denaturing SDS-PAGE indicated a molecular mass of 60 kDa. Thus, from these data we suggest that lettuce polyphenol oxidase is a tetramer of identical subunits.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Purification and characterization of a mannan-binding lectin specifically expressed in corms of saffron plant (Crocus sativus L.)

Despite the economical interest of Crocus sativus, its biochemistry has been poorly studied. Herein, we have isolated a lectin present in saffron corm by gel-filtration, anion-exchange, and reversed-phase chromatography. One- and two-dimensional PAGE, MALDI-MS, and N-terminal amino acid sequence analyses indicated that the native protein forms noncovalently linked aggregates of about 80 kDa apparent molecular mass, mainly composed of two charged heterogeneous (pI's, 6.69-6.93) basic subunits of approximately 12 kDa. Their N-terminal sequences shared 25% similarity and were homologous to the N- and C-terminal domains of monocotyledonous mannose-binding lectins, respectively. An additional polypeptide of around 28 kDa apparent molecular mass was also detected, probably corresponding to a precursor processed into two mature subunits. In addition, the N-terminal domain subunit exhibited 56% similarity with curculin, a sweet protein with taste-modifying activity. The native lectin specifically interacts with a yeast mannan and is a major corm protein specifically expressed in this organ.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

New mannose-binding lectin isolated from the rhizome of Sarsaparilla Smilax glabra Roxb. (Liliaceae)

A new mannose-binding lectin, designated SGM2, was isolated from the rhizome of a Chinese medicinal herb Smilax glabra (also known as sarsaparilla in general) by saline extraction, ammonium sulfate precipitation and fractionation, and affinity chromatography on fetuin- and mannose-agarose. SGM2 is shown to have a molecular mass of 37 kDa on gel filtration and 12.5 kDa on SDS-PAGE, indicating that it is a trimeric protein composed of three identical subunits. When the first 30 amino acid residues at the N-terminal were compared, SGM2 had approximately 40% homology with those of some other monocots. SGM2 had the property of hemagglutinating activity toward rabbit erythrocytes, which could be reversed by mannose and mannose polymers. SGM2 exhibited antiviral activities against both herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV) with the same EC(50) of 8.1 microM.     

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Characterization of the proteins from Vigna unguiculata seeds

The proteins from Vigna unguiculata (L.) Walp. (cowpea) seeds were investigated. Globulins constitute over 51% of the total seed protein, with albumins composing approximately 45%. The globulins may be fractionated by native electrophoresis or anion exchange chromatography into three main components, which were termed (in decreasing order of anodic mobility) alpha-vignin, beta-vignin, and gamma-vignin. alpha-Vignin, with a sedimentation coefficient of 16.5S, is a major, nonglycosylated globulin, composed of a major 80 kDa subunit, which upon reduction, produces two polypeptides (20 and 60 kDa). beta-Vignin, with a sedimentation coefficient of 13S, is a major, glycosylated globulin, composed of two main polypeptides (55 and 60 kDa) with no disulfide bonds. Finally, gamma-vignin, a minor globulin, is composed by one main type of subunit (22 kDa), which upon reduction, is converted into a single, apparently heavier polypeptide chain (30 kDa) due to the presence of an internal disulfide bond. Immunological analyses revealed structural homology between beta-vignin and beta-conglutin (the vicilin from Lupinus seeds) but not between alpha- or gamma-vignins and their Lupinus counterparts. Haemagglutination activity toward trypsinized rabbit erythrocytes was found exclusively in the albumin fraction and was strongly inhibited by N-acetylglucosamine or chitin.     

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Beta-glucosidase from Chalara paradoxa CH32: purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degrees C. The enzyme was inactivated at 60 degrees C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg(2+) and Ag(2+) and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The V(max) of the enzyme for p-NPG (K(m) = 0.52 mM) and cellobiose (K(m) = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K(i) = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.     

Purification and characterization of a beta-glucosidase from Citrus sinensis var. Valencia fruit tissue

A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.     

Lectin from Phaseolus acutifolius var. escumite: chemical characterization, sugar specificity, and effect on human T-lymphocytes

Purification of the lectin from Phaseolus acutifolius var. escumite was achieved by affinity chromatography on a column containing glutaraldehyzed membranes from blood group O erythrocytes. The lectin is a tetrameric glycoprotein of 121 kDa with 10% of sugar by weight composed by four subunits of 30 kDa as determined by SDS-PAGE. The lectin is composed of four isolectins as determined by ion-exchange chromatography on a mono-S column. The lectin and its isolectins showed identical NH2 terminal residues (ANDLSFNFQR FNETN) with homology to the PHA leucoagglutinin-precursor. Peptide mass fingerprint from each lectin isoform determined from tryptic peptides by MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) showed differences among subunits, thus suggesting microheterogeneity in their amino acid sequences or different glycosylation patterns. The lectin and its four isolectins agglutinated erythrocytes without serological specificity and showed mitogenic activity on human leukocytes; moreover, the main effect was rather toward CD8+ than to CD4+ human peripheral lymphocytes. The lectin from escumite was not inhibitable by simple sugars; however, the specificity of the lectin and its isoforms was mainly addressed toward galactose residues present in bi- or triantennary N-acetyllactosamine-type glycans.     

Are molecular weights of proteins determined by superose 12 column chromatography correct?

Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.     

Larvicidal effects of a chitin-binding vicilin from Erythrina velutina seeds on the mediterranean fruit fly Ceratitis capitata

Chitin-binding vicilin from Erythrina velutina seeds was purified by ammonium sulfate followed by affinity chromatography on a chitin column and gel filtration on Superose-6-10-300-GL. The Erythrina velutina vicilin, called EvV, is a tetrameric glycoprotein composed of 1.85% carbohydrates and M r of 216.6 kDa, consisting of two subunits of M r of 54.8 and two subunits of M r of 50.8 kDa. The EvV homogeneity was confirmed in native PAGE where it was observed to be a unique acid-protein band with slow mobility in this gel. Effect of EvV on C. capitata larvae was examined by bioassay and its mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV when added to diet caused strong effect on mortality (ED50 of 0.14%) and larval mass (WD50 of 0.12%). These deleterious effects were associated to the binding to chitin structures present in peritrophic membrane and to gut epithelial cells, and its low digestibility in C. capitata digestive tract. These results are the first demonstration of a proteinaceous bioinsecticide from plant origin effective against C. capitata larvae. EvV may be part of the pest management programs or an alternative in plant improvement program.     

Analysis of the lectins from teosinte (Zea diploperennis) and maize (Zea mays) coleoptiles

To identify molecular evidence of the common origin of maize and teosinte, a lectin from teosinte coleoptile (TCL) was purified, through affinity chromatography on a lactosyl-Sepharose column, and some of the physicochemical parameters were compared with those from the maize coleoptile lectin (CCL). TCL is a 92 kDa glycoprotein constituted mainly by aspartic, glutamic, glycine, leucine, and lysine residues; in minor proportion, methionine and cysteine were also found. The glycannic portion of the lectin, which corresponds to 10% w/w, is composed by Gal, Man, and GlcNAc. CCL is an 88.7 kDa glycoprotein that contains 12% sugars by weight; its sugar and amino acid compositions are similar to those of TCL. TCL is formed by two isoforms identified through acidic electrophoresis, whereas CCL is constituted by a single molecular form. The NH(2) termini of both TCL isoforms are blocked, but their amino acid sequences determined from tryptic peptides by matrix-assisted laser desorption ionization time-of-flight) indicated that TCL isoforms have no homology with other mono- or dicotyledonous lectins, including CCL. TCL, just as CCL, showed hemagglutinating activity toward animal erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that although TCL shows broader sugar specificity than CCL, it recognizes Gal in O- and N-glycosidically linked glycans. Both lectins are equally well recognized by antibodies against TCL.     

Elucidation of the emulsification properties of sugar beet pectin

A protocol has been developed to fractionate sugar beet pectin using hydrophobic affinity chromatography. Three samples eluted from the column using 4 M NaCl as solvent (fractions 1A, 1B, and 1C), two fractions eluted using 2 M NaCl (fractions 2A and 2B), and one fraction eluted using water (fraction 3). The fractions were shown to be very polydisperse, and differences between the GPC refractive index and UV absorbance (214 nm) elution profiles demonstrated chemical heterogeneity. They were found to contain significantly different proportions of protein (1A, 2.79%; 1B, 0.97%; 1C, 0.77%; 2A, 1.41%; 2B, 5.09%; and 3, 5.89%) and ferulic acid (approximately 1A, 0.5%; 1B, 0.5%; 1C, 0.9%; 2B, 1.5%; and 3, 2%). The weight-average molecular mass, M(w), of the fractions also varied (1A, 153 kDa; 1B, 155 kDa; 1C, 306 kDa; 2A, 562 kDa; 2B, 470 kDa; 3, 282 kDa). Three fractions, that is, 1A, 1B, and 3, produced orange oil emulsions with a relatively small droplet size that were stable over a period of weeks. The other three fractions (1C, 2A, and 2B with higher M(w) values) produced emulsions with an initially larger droplet size, and the droplet size increased considerably over time. The increased droplet size may be influenced by the viscosity of the aqueous continuous phase. There was no simple relationship between protein or ferulic acid content and emulsification ability. For example, fraction 1B, which contained the lowest proportion of both protein and ferulic acid, produced stable emulsions of similar droplet size to fraction 3, which contained the largest proportion of protein and ferulic acid. The role of protein in the emulsification process was investigated by measuring the amount of protein in the aqueous phase before and after emulsification. It was clearly demonstrated that proteinaceous material adsorbed at the oil-water interface. It is evident that the emulsification properties of sugar beet pectin are influenced by the accessibility of the protein and ferulic acid groups to the surface of the oil droplets, the proportion of ester groups, and the molecular mass distribution of the fractions.     

Isolation of isolectins from Vitis vinifera L. Cv. Chardonnay grape berries

A lectin fraction from Chardonnay grape juice has been isolated by affinity chromatography on a column of p-aminophenyl beta-D-glucoside-derivatized agarose. The lectin fractions agglutinate rabbit and human erythrocytes without serological specificity. None of the usual monosaccharides, glycosides, or glycoproteins inhibit the hemagglutinating activity. Erythroagglutination is only inhibited by nitrophenyl glycosides, p-nitrophenyl beta-D-glucoside being the strongest inhibitor. In SDS-PAGE in the presence of 2-mercaptoethanol and gel filtration HPLC, the lectin fraction gave a single band or peak corresponding to M(r) 13.2-11.9 kDa, thus indicating it to be a monomer. Three bands were observed by isoelectric focusing with pI values of 4.1, 4. 4, and 4.9. The isolectins seem to be glycoproteins since they are bound on a concanavalin A-Sepharose column.     

Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening

The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi.     

Chemical characterization of the high molecular weight material extracted with hot water from green and roasted arabica coffee

The polysaccharides present in coffee infusions are known to contribute to the organoleptic characteristics of the drink, such as the creamy sensation perceived in the mouth known as "body", the release of aroma substances, and the stability of espresso coffee foam. To increase the knowledge about the origin, composition, and structure of the polysaccharide fraction, the high molecular weight material (HMWM) was extracted with hot water from two green and roasted ground arabica coffees: Costa Rica (wet processed) and Brazil (dry processed). The polysaccharides present in the green coffees HMWM were arabinogalactans (62%), galactomannans (24%), and glucans, and those found in roasted coffees were galactomannans (69%) and arabinogalactans (28%). The polysaccharides of the HMWM of the roasted coffees were less branched than those of the green coffees. The major green coffee proteins had molecular weights of 58 and 38 kDa, and the 58 kDa protein had two subunits, of 38 and 20 kDa, possibly linked by disulfide bonds. The protein fraction obtained from roasted coffees had only a defined band with < or =14 kDa and a diffuse band with >200 kDa. The majority of the galactomannans were precipitated with solutions of 50% ethanol, and the size-exclusion chromatography of the roasted fractions showed coelution of polysaccharides, proteins, phenolics, and brown compounds. The use of strong hydrogen and hydrophobic dissociation conditions allowed us to conclude that the phenolics and brown compounds were linked by covalent bonds to the polymeric material.