Basic Protein Fraction Research Articles

Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.

Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. To analyse the allergenic composition of the basic peanut protein (BPP) fraction. A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. Most IgE reactivity of the BPP fraction was due to the 5-7kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.

Electrophoretic system for express analysis of whey protein fractions

Fractional specificity of biological action and ability to form bioactive peptides, which have a positive effect on different physiological systems of the body in the processes of proteolysis and digestion, are characteristic for whey proteins. Prospects for the production and application of whey protein fractions are related to the necessity of their composition control. The comparative analysis of the electrophoretic systems previously used for the milk protein analysis was carried out for the creation of an express analysis method of whey protein fractions. These are the anode disc electrophoresis system in the presence of sodium dodecyl sulfate, the Davis disc electrophoresis system for acidic proteins in native conditions, the system in a homogeneous polyacrylamide gel with urea. The Davis disc electrophoresis system for acidic proteins was chosen as the basis. For the adaptation of this system to the requirements of express analysis, the stacking polyacrylamide gel was removed from its composition and the concentration of the separating gel was reduced. The difference in the composition of electrode buffer and gel buffer ions was used to provide the high separation efficiency of protein fractions. This allows saving the effect of protein concentration in the whey sample on the first stages of electrophoresis. The position of the basic whey protein fractions on electrophoregrams was established with the help of homogeneous marker proteins (β-lactoglobulin and whey albumin). Аn accessible electrophoresis system in the slabs of a homogeneous polyacrylamide gel for serial express analysis of the fractional composition of whey proteins has been proposed as a result of researches. The system allows reliable identification of four protein fractions (α-LA, β-LG, BSA and IG). Close average values and standard deviations of the content of these fractions in 15 whey samples of one milk batch, obtained by the densitometry of three electrophoregrams: β-LG (37.3±4.2, 36.5±2.8; 38.3±2.7), α-LA (15.8±1.5, 15.8±1.3, 16.4±1.1), BSA (8.2±1.1, 8.0±1.0, 9.4±1.1), IG (17.6±1.9, 17.4±1.5, 16.8±1.5) testify about good reproducibility of the method. The proposed method may be useful for the express identification of the basic whey protein fractions, which are precursors of biologically active peptides.

Impact of Whey Protein on Bone Mineral Density: a Systemic Review and Meta-analysis

Introduction Previous studies have shown that whey protein and its basic protein fraction, milk basic protein, might have a beneficial effect on bone mass through promoting bone formation and suppressing osteoclast activity. Nonetheless, most studies do not have enough power to examine the potential benefit on bone mass from using whey protein and milk basic protein and the data are contradictory. Objective The aim of our meta-analysis is to reveal the impact of whey protein and milk basic protein on bone mineral density (BMD). Method We comprehensively searched the databases of MEDLINE, EMBASE, and Cochrane databases. The inclusion criterion was published randomized control trials (RCT) comparing whey protein supplementation or milk basic protein to placebo or controls (non-whey protein). The primary outcome was the differences in the changes in BMD at the level of lumbar spine, hip, or femoral neck, which was primarily measured by dual-energy x-ray absorptiometry. We calculated pooled mean difference (MD) with 95% confidence intervals (CI) using random-effects model. Publication bias was assessed using funnel plot and Egger's regression test Results Six RCTs were included in the meta-analysis involving 562 subjects. Our collected studies utilized varying dose of whey protein from 25 grams to 75 grams per day and the dose of milk basic protein is 40 grams per day during a time frame ranging from six months to two years. There was a statistically significant improvement of BMD at the lumbar spine (MD= 0.014, 95% CI: 0.011 to 0.017, p-value Conclusion This is the first meta-analysis investigating the impact of whey protein and milk basic protein on BMD. Our study suggested that whey protein supplementation and milk basic protein, the basic protein fraction of whey protein, might have a positive impact on bone mineral density compared to placebo or controls. Using whey protein and milk basic protein should be considered to be a conjunctive treatment with current therapy to improve bone mineral density.

Influence of micellar calcium and phosphorus on rennet coagulation properties of cows milk

The main requirement for milk processed in most cheese typologies is its rennet coagulation ability. Despite the increasing number of studies, the causes for abnormal coagulation of milk are not fully understood. The aim of this study was to ascertain relationships between milk characteristics and its rennet coagulation ability, focusing on the influence of calcium (Ca) and phosphorus (P). Ca and P are essential constituents of the micelles. Micellar P can be present as part of colloidal calcium phosphate (inorganic-P) or covalently bound to caseins as phosphate groups (casein-P). Eighty one herd milk samples (SCC<400000cell/ml) were classified as Optimal (8), Suboptimal (39) Poor (29) and Non-coagulating milk (5), according to their rennet coagulation parameters as assessed by lactodynamographic test. Samples were analysed for their chemical composition (basic composition, protein fractions, minerals and salt equilibria), physicochemical parameters (pH and titratable acidity) and rheological properties. Optimal milk was characterised by the highest contents of major constituents, protein fractions and minerals, lowest content of chloride and highest values of titratable acidity. Non-coagulating milk was characterised by the highest values of pH and the lowest of titratable acidity. At micellar level, Optimal milk showed the highest values of colloidal Ca, casein-P and colloidal Mg (g/100g casein), while Non-coagulating milk showed the lowest values. Interestingly, there was no statistical difference regarding the content of colloidal inorganic-P (g/100g casein) between Optimal and Non-coagulating milks. Overall, high mineralisation of the micelle (expressed as g inorganic-P/100g casein) positively affect its rennetability. However, excessive mineralisation could lead to a reduction of the phosphate groups (g casein-P/100g casein) available for curd formation.

Evaluation of milk basic protein supplementation on bone density and bone metabolism in Chinese young women

Milk is a good source of bioavailable calcium compared with other foods. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The objective of this study was to examine the effects of MBP on bone mineral density (BMD) and bone metabolism of healthy young women. Eighty-four healthy young women were randomly assigned to three groups: control group, whole milk group or MBP group treated with milk containing 40 mg MBP for 8 months. The bone mineral density of total body, the lumbar vertebrae L2-L4 and the left forearm of each subject were measured by dual-energy X-ray absorptiometry (DEXA) at 0 and 8 months of treatment. Serum indexes of bone metabolism were measured at 0, 3, 6 and 8 months. Eighty-one subjects who completed the study in accordance with the protocol were included in the analysis. Total BMD in all groups significantly increased compared with baseline values. However, no significant difference on the mean rate of gain of total BMD was observed among the MBP group (2.19%), the whole milk group (2.63%) and the control group (1.61%). Serum cross-linked N-teleopeptides of type-I collagen (NTx) in MBP group at 8 months and in whole milk group at 6 months were significantly decreased from baseline. There were no significant differences between whole milk group and MBP group; however, after combining the milk groups, NTx had significantly decreased from baseline. No significant increase was observed in serum bone-specific alkaline phosphatase (BAP) in both whole milk group and MBP group. No significant effect of MBP on bone mineral density and bone metabolism was observed, but milk supplementation was effective in suppressing bone resorption.

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.

Assessment of the potential allergenicity of a Milk Basic Protein fraction

Background A specific basic fraction of bovine milk, termed Milk Basic Protein (MBP), has the potential to provide nutritionally important benefits if used as a food ingredient. Although derived from milk, MBP is intended for use as an ingredient in other foods. Cows’ milk is a well studied, commonly allergenic food. Although the proteins in MBP are not identified as milk allergens, food products containing MBP will be labelled as containing milk as a caution to milk allergic consumers under food labelling guidelines in the US and the European Union as MBP has not been demonstrated to be free of milk allergens. However, as part of an overall safety evaluation of MBP, the developers sought to evaluate the potential allergenicity of the primary protein components for characteristics of allergenic food proteins and to assess whether intake of these proteins at intended use levels could present a significant new allergenic risk for consumers. Objective To evaluate the potential allergenicity of the five identified proteins in MBP. While extensive studies have not demonstrated allergenicity of lactoferrin, the four other proteins are less studied. The four were tested here by sequence identity comparison to known allergens, and for stability of these proteins in acidic pepsin as a characteristic common to many food allergens. Methods Sequences of the proteins were compared to those listed in AllergenOnline.com, by methods recommended for the evaluation of proteins introduced in crops through genetic engineering. Pepsin stability was assessed by incubating the various proteins in simulated gastric fluid at pH 1.2 with porcine pepsin for up to 60 min at 37 °C, with samples withdrawn and analyzed at specific times. Results No significant sequence similarities were identified for the MBP proteins compared to known allergens. All but one of the protein components of MBP were digested relatively quickly by pepsin. The more stable protein will be of low abundance as consumed in contrast to most pepsin-stable food allergens. Conclusions Based on molecular characteristics and expected exposure, the protein components in MBP are unlikely to present any increased risk of allergy for milk allergic subjects or of cross-reactivity for other allergic subjects. However, since the proteins are derived from milk, products containing MBP will need to be labelled as containing milk proteins to warn milk allergic subjects of the potential risk of allergic reactions.

Safety evaluation of a milk basic protein fraction

Milk products are widely consumed by individuals in the US population in the form of fluid milk and milk-derived products and ingredients. Milk is a good source of calcium, which plays a role in maintaining bone health. In addition to calcium, the whey protein fraction of milk contains basic proteins that have been demonstrated to increase bone metabolism and inhibit bone resorption. A specific basic protein fraction in milk (Milk Basic Protein; MBP) was tested in an acute oral toxicity study, teratology study, subchronic oral toxicity study, and reverse mutation assay and no treatment related adverse effects were found. MBP has been evaluated for its use as an ingredient in food and concluded to be safe for its intended use.

The Fragments of Bovine High Molecular Weight Kininogen Promote Osteoblast Proliferation In Vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.

Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women

Effect of milk basic protein on bone metabolism in healthy young women. Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone mineral density and bone metabolism of healthy young women. Thirty-five healthy young women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indexes of bone metabolism were measured at 0, 3 and 6 months. All subjects completed the study in accordance with the protocol. The mean rate of gain of lumbar BMD in the MBP group (1.57%) was significantly higher than in the placebo group (0.13%, P=0.042). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased, and serum osteocalcin was significantly increased in the MBP group at 6 months. These results suggested that MBP supplementation was effective in increasing BMD in young women and that this increase in BMD may be primarily mediated through the promotion of bone formation and inhibition of bone resorption by MBP supplementation.

Prevention of osteoporosis by foods and dietary supplements. Bone reinforcement factor in milk: milk basic protein (MBP)

Milk has been drunk widely for long time, because it contains superior nutritive value. In particular, milk is the supplying source of good calcium of bioavailability, in comparison with other foods. In addition, whey protein is a by-product of cheese and casein production, and we have showed that this whey protein plays a functional role for bone remodeling (bone formation and bone resorption). And, it became clear by an examination of in vitro that there were the active substances that promote bone formation and suppress bone resorption in the basic protein fraction (milk basic protein: MBP). Furthermore, it was shown that whey protein and MBP enhanced bone strength of femur in ovariectomized rats. In addition, some human studies clearly showed that MBP improved the balance of bone metabolism and increased bone density. As for MBP, MBP, which contributes to bone health, is expected as a new food material extracted from milk.

Prevention of osteoporosis by foods and dietary supplements. Milk basic protein (MBP) increases bone mineral density in young adult women and perimenopausal women

Milk has beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein: MBP), contains components capable of promoting bone formation and inhibiting bone resorption. We tried to examine the effect of MBP on bone mineral density (BMD) and markers of bone metabolism in healthy young adult women and perimenopausal women. In the healthy young women study, we found that MBP increased BMD, by promoting bone formation and inhibiting bone resorption. In the healthy perimenopausal women study, we also found that MBP increased BMD, primarily by inhibiting bone resorption, while maintaining bone formation. Thus, bone remodeling balances become positive that leads to maintenance or increase of bone formation. MBP supplementation could be effective for bone health in a wide range of generation especially those who hate to drink milk.

A comparison between acidic and basic protein fractions from whey or milk for reduction of bone loss in the ovariectomised rat

The ability of whey acidic protein fractions to protect against bone loss due to ovariectomy (OVX) in the mature female rat was investigated. The bone bioactivity of these acidic protein fractions, isolated from both mineral acid whey protein concentrate (WPC) and rennet WPC, was compared with that of basic protein fractions isolated from both milk and rennet WPC. Fifty 6-month old rats that had been ovariectomised at 5.5 months were randomised into five groups of ten each. One group remained the OVX control while four groups were each fed one of the acidic or basic protein fractions as 0.3% (w/w) of the diet, for 4 months. Ten sham-operated rats served as a second control group. Sequential measurements of bone mineral density of the spine and femur indicated that the acidic protein fraction prepared from mineral acid WPC reduced bone loss due to OVX, maintaining bone density above OVX levels at week 16 of feeding. Biomechanical data indicated that both acidic fractions tended to increase bone stiffness, and hence resistance against breaking.

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC 50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml −1) and by HeLa, HT29 and JM cells with IC 50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml −1, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.

A controlled trial of the effect of milk basic protein (MBP) supplementation on bone metabolism in healthy menopausal women

Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone metabolism of healthy menopausal women. Thirty-two healthy menopausal women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indices of bone metabolism were measured at 0, 3 and 6 months. Twenty-seven subjects who completed the study in accordance with the protocol were included in the analysis. The mean rate of gain of lumbar BMD in the MBP group (1.21%) was significantly higher than in the placebo group (-0.66%, P=0.046). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased in the MBP group at 6 months, but no significant difference in serum osteocalcin was observed between the two groups. The urinary NTx excretion was found to be related to serum osteocalcin in the MBP group at 3 and 6 months, indicating that MBP maintained the balance of bone remodeling. These results suggested that MBP supplementation was effective in preventing bone loss in menopausal women and that this improvement in BMD may be primarily mediated through the inhibition of bone resorption while maintaining the balance of bone remodeling by MBP supplementation.

The effect of whey acidic protein fractions on bone loss in the ovariectomised rat

Bovine milk has been shown to contain bioactive components with bone-protective properties. Earlier studies on bovine milk whey protein showed that it suppressed bone resorption in the female ovariectomised rat. A new osteotropic component was subsequently identified in the whey basic protein fraction, but bone bioactivity may also be associated with other whey fractions. In the present study, we investigated whether acidic protein fractions isolated from bovine milk whey could prevent bone loss in mature ovariectomised female rats. Six-month-old female rats were ovariectomised (OVX) or left intact (sham). The OVX rats were randomised into four groups. One group remained the control (OVX), whereas three groups were fed various whey acidic protein fractions from milk whey as 3 g/kg diet for 4 months. Outcomes were bone mineral density, bone biomechanics and markers of bone turnover. Bone mineral density of the femurs indicated that one of the whey AF over time caused a recovery of bone lost from OVX. Plasma C-telopeptide of type I collagen decreased significantly in all groups except OVX control over time, indicating an anti-resorptive effect of whey acidic protein. Biomechanical data showed that the AF may affect bone architecture as elasticity was increased by one of the whey AF. The femurs of AF-supplemented rats all showed an increase in organic matter. This is the first report of an acidic whey protein fraction isolated from milk whey that may support the recovery of bone loss in vivo.

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 μg(protein) cm−3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl2. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.

Physicochemical and kinetic properties of unique isozymes of UDP-Glc pyrophosphorylase that are associated with resistance to sweetening in cold-stored potato tubers

Isozymes of UGPase with unique catalytic properties were purified from the cold-induced-sweetening (CIS) resistant cultivar Snowden (Solanum tuberosum). Two distinct peaks of UGPase activity were obtained when protein extracts were subjected to anion-exchange chromatography on DEAE-Sephacel. Polypeptides in the first eluted fraction (A-I) were ionically similar to the UGPase isozyme UGP3 previously purified and characterized from the cold-sweetening sensitive cultivar Norchip (Sowokinos et al. 1993, Plant Physiol 101: 1073-1080). Seventy-two percent of the total endogenous UGPase activity in Snowden (cv.) tubers, however, was found in a more basic protein fraction (A-II) that is not found in the Norchip cultivar. This study reports on the physicochemical and kinetic properties of these new polypeptides that demonstrate UGPase activity. The reaction in the direction of UDP-Glc synthesis was specific for the substrates Glc-1-P and UTP and there was an absolute requirement for Mg2+ ions. The catalytic properties of UGP5 were markedly different from UGPase isozymes previously described in terms of (1) affinity for the substrate Glc-1-P, (2) pH optimum, (3) maximum reaction velocity and (4) sensitivity to product inhibition with UDP-Glc. Chi-square analysis of fifty-four genetically diverse potato lines revealed that resistance to CIS was highly correlated with the presence of the A-II isozymes of UGPase. The kinetic properties of these unique forms of UGPase may underlie, in part, a tuber's ability to resist sweetening in the cold.

An organotypic culture of the newt retina together with other tissues of the posterior eye segment as a model for studying the effects of the cell adhesion glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins and the localization of acetylcholinesterase activity were found to appear during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.

Evidence of an endogenous lectin receptor in seeds of the legume Cratylia floribunda

Cratylia floribunda seeds were ground and the clean crude saline extract was fractionated into albumin, globulin, prolamin, acidic and basic glutelin protein fractions. These protein fractions were examined for the presence of an endogenous lectin receptor by SDS-polyacrylamide gel electrophoresis, western blot, affinity chromatography on a Sepharose 4B-Cratylia floribunda (CFL) lectin column and kinetic analysis in real time by surface plasmon resonance (SPR). Prolamin was the richest protein fraction although very poor in haemagglutinating activity. Basic glutelin was far the less interesting fraction for lectin activity and protein content, even though this fraction contains considerable amounts of carbohydrates. Lectin was present in all protein fractions as estimated by haemagglutinating assays but basic glutelins were almost devoid of lectin activity. Except for prolamins, protein bands were detected by SDS-PAGE in all other fractions. Western blot using digoxigenin labelled Con A revealed a single band in the albumin, globulin, acidic and basic glutelin fractions, which specifically interacted with ConA. This band migrated exactly at the same position in such fractions and seemed to be more important in the globulins. Affinity chromatography of the protein fractions on a Sepharose-CFL column yielded a peak, which was only recovered after elution with acidic buffered solution or with an alpha-D-mannose solution and the monosaccharide was recognized by the lectin. These results were fully corroborated by real time interaction of immobilized CFL with the different soluble protein fractions suggesting the presence of a lectin receptor within albumins, globulins and basic glutelins. As a whole, the results suggest that the lectin from Cratylia floribunda recognizes a soluble endogenous glycosylated receptor through an interaction mediated by its carbohydrate-binding site.