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PubMed1975

Electrophoresis of cottage cheese whey proteins and their polymers.

Cottage cheese whey solutions containing sodium dodecyl sulfate were resolved electrophoretically on 5% sodium dodecyl sulfate polyacrylamide gels. Major bands were identified by comparing their electrophoretic mobilities to those of known whey components. Other components were identified principally from molecular weights determined from a calibration correlating mobility and molecular weight. Conditions which affect polymerization of proteins were studied in whey and solutions of purified beta-lactoglobulin. Formation of a number of polymers was induced by concentrating the whey samples, lowering the temperature, adjusting pH, or adding salts. The dimer, trimer, tetramer, and octamer of beta-lactoglobulin, the dimer and trimer of bovine serum albumin, and several unidentified components in the 100,000 to 300,000 molecular weight range were observed. The octamer state of beta-lactoglobulin was observed in whey at pH values between 5.1 and 8.0, at temperatures below 10 C, and with .2M addition of potassium thiocyanate, potassium iodide, calcium chloride, or sodium acetate. Similar polymer formation, and temperature and pH effects, were observed with solutions of purified beta-lactoglobulin, which contained dimers, trimers, and tetramers. The beta-lactoglobulin octamer in whey samples could be dissociated by the addition of acid. The bovine serum albumin dimer appeared in whey at pH above 6.2 and at -minus 1 C.

Animals↗
PubMed1975

Composition of milks of dairy cattle. I. Protein, lactose, and fat contents and distribution of protein fraction.

Milks from commercial dairy herds in Southeastern Pennsylvania were analyzed for total protein, casein, whey protein, beta-lactoglobulin, nonprotein nitrogen, and lactose contents. Data for fat contents and milk yields were from Dairy Herd Improvement Association records for the same lactation. Milk samples were from a single milking of healthy cows (151) in midlactation. Since the remainder of the milk was returned to the bulk milk of the farm, the data represent market milk composition. The data were grouped and analyzed by breed and beta-lactoglobulin phenotype; there were 18 to 33 cows per breed. In true protein percentage, the breeds ranked: Jersey 4.07 plus or minus .49, Brown Swiss 3.84 plus or minus .47, Guernsey 3.56 plus or minus .53, Ayrshire 3.30 plus or minus .52, Milking Shorthorn 3.17 plus or minus .47, Holstein 3.07 plus or minus .43. Breeds differed in all other components and in milk yield. Brown Swiss ranked highest in yield of protein. Only whey protein and beta-lactoglobulin contents were influenced by the beta-lactoglobulin genotype with beta-lactoglobulin A greater than AB greater than B in whey protein content.

Animals↗
PubMed1980

Passage of ingested protein into the blood during gastrointestinal hypersensitivity reactions: experiments in the preruminant calf.

Preruminant calves given a series of feeds of heated soya bean flour (HSF) frequently develop gastrointestinal hypersensitivity to soya bean antigens. The permeability of the intestinal mucosa of calves undergoing hypersensitivity reactions to ingested HSF has been assessed by feeding them milk and quantitating the leakage of beta-lactoglobulin into the blood. Maternal beta-lactoglobulin did not evoke an antibody response in calves. Therefore its detection in the serum was not influenced by immunological mechanisms that normally remove or exclude antigens from the circulation. In sensitized calves ingestion of HSF caused a dramatic increase in the permeability of the intestine to beta-lactoglobulin. The change was transitory and after 24 hr permeability had almost returned to normal. The mucosal barrier was not permanently damaged regardless of the number or the severity of the reactions experienced. Indomethacin was ineffective in counteracting permeability changes. A progressive increase in the sensitivity of the gut to soya bean antigens was accompanied by a rise in the titre of serum antibodies to soya bean proteins. Absorbed beta-lactoglobulin was present in the serum in its monomeric form only, and quickly disappeared from the circulation. In an enzyme immunoassay used to measure its concentration absorbed beta-lactoglobulin was indistinguishable from the native protein. These results suggest that measurement of intestinal permeability to macromolecules might be useful in the diagnosis of certain forms of food allergy in man.

Animals↗
PubMed1993

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes.

1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established. 2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets. 3. An inhibitory effect of bovine alpha-lactalbumin and of beta-lactoglobulin on lactoferrin-receptor interaction was shown. 4. Bovine alpha-lactalbumin competes with lactoferrin for the binding sites. 5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of alpha-lactalbumin with an affinity constant, Ka = 0.46 x 10(9) mol/l and 335 receptors/cell. 6. The inhibitory effect of beta-lactoglobulin reaches 62% and is different for the common fraction beta-lactoglobulin and the genetic variants beta-lactoglobulin A and B. 7. beta-lactoglobulin does not compete with lactoferrin for the membrane receptors. 8. Bovine casein and egg lysozyme stimulate 59Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown. 9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures. 10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

Animals↗
PubMedDate not supplied

Mechanical characterization of network formation during heat-induced gelation of whey protein dispersions.

The formation of gel network structures during isothermal heating of whey protein aqueous dispersions was probed by mechanical spectroscopy. It was anticipated that the pathway of the sol-to-gel transition of whey protein dispersions is quite different from that of ordinary cross-linking polymers (e.g., percolation-type transition), since aqueous solutions of native whey proteins have been shown to be highly structured even before gelation, in our previous study. At 20 degrees C, aqueous dispersions of beta-lactoglobulin, the major whey protein, and those of whey protein isolate (WPI), a mixture of whey proteins, exhibited solid-like mechanical spectra, i.e., the predominant storage modulus G' over the loss modulus G", in a certain range of the frequency omega (1-100 rad/s), regardless of the presence or absence of added NaCl. The existence of the added salt was, however, a critical factor for determining transitions in mechanical spectra during gelation at 70 degrees C. beta-Lactoglobulin dispersions in 0.1 mol/dm(3) NaCl maintained the solid-like nature during the entire gelation process and, after passing through the gelation point, satisfied parallel power laws (G' approximately G" approximately omega(n)) that have been proposed for a critical gel (i.e., the gel at the gelation point) that possesses a self-similar or fractal network structure. In contrast, beta-lactoglobulin dispersions without added salt exhibited a transition from solid-like [G'(omega) > G"(omega)] to liquid-like [G'(omega) < G"(omega)] mechanical spectra before gelation, but no parallel power law behavior was recognized at the gelation point. During extended heating time (aging), beta-lactoglobulin gels with 0.1 mol/dm(3) NaCl showed deviations from the parallel power laws, while spectra of gels without added NaCl approached the parallel power laws, suggesting that post-gelation reactions also significantly affect gel network structures. A percolation-type sol-to-gel transition was found only for WPI dispersions without added salt.

Animals↗
PubMedDate not supplied

Increased serum antibody levels against cow's milk proteins in children with chronic liver disease.

We measured IgG, IgM and IgA antibodies to cow's milk proteins: alpha-casein, alpha-lactalbumin, bovine serum albumin, beta-lactoglobulin-a and beta-lactoglobulin-b in the sera of 29 pediatric patients with liver disease. IgG antibodies to bovine serum albumin, beta-lactoglobulin-a and beta-lactoglobulin-b and IgA antibodies to alpha-casein were elevated in most of the patients compared to age-matched controls. We postulate that, in patients with liver disease either increased intestinal uptake resulting in increased humoral response, or decreased hepatic clearance of food antigens intensifying a humoral response to these foreign antigens, may occur.

Adolescent↗
PubMed2003

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.

Antibodies↗
PubMed2002

Kinetics of disproportionation of air bubbles beneath a planar air-water interface stabilized by food proteins.

The rate of shrinkage of air bubbles of initial radii, r, from 50 to 150 microm injected beneath a planar air-water interface has been measured. Bubbles were stabilized by 0.05 wt% protein in approximately 0.1 mol dm(-3) ionic strength buffer at pH 7.0 and at room temperature. Four proteins were studied: commercial whey protein isolate (WPI), sodium caseinate, gelatin, and pure beta-lactoglobulin. Bubbles in all systems showed shrinkage due to diffusion of gas from the bubbles, which accelerated as the bubbles got smaller. Within approximately 1 h all bubbles had disappeared, having shrunk to below approximately 1 microm, so that in no cases was there evidence of stabilization via a surface rheological mechanism. The rates of shrinkage with the different proteins were not significantly different except in the case of gelatin, which at any given bubble size appeared to give a slightly higher rate, probably because the surface tension is higher for this system. A new theoretical analysis of the dissolution kinetics for the case of a bubble close to a planar interface has been developed. For caseinate and WPI a simple model incorporating a constant surface tension and a constant bubble-interface separation appears to account for the kinetics. Interestingly, the model predicts a linear dependence of r(n) versus time when n is closest to 3, in contrast to n = 2 expected from previous work. For gelatin and pure beta-lactoglobulin, the introduction of modest dilatational elasticities of approximately 2.3 and 7 mN m(-1), respectively, gives good agreement between theory and experiment. This is particularly the case for beta-lactoglobulin, where there is a noticeable slowing, but not cessation, of the shrinkage as the bubbles get smaller. In the light of these findings the practical significance of surface rheology with respect to stability to disproportionation is discussed. Finally, we present experimental evidence that a bubble stabilized by beta-lactoglobulin shrinks to a nonspherical protein particle consisting of the completely collapsed protein film.

Adsorption↗
PubMed1992

Comparison of the intestinal and serum antibody response in patients with dermatitis herpetiformis.

Dermatitis herpetiformis (DH) is an intensely pruritic, blistering skin disease characterized by cutaneous IgA deposits and an associated, most often asymptomatic, gluten-sensitive enteropathy. When patients with DH are placed on a gluten-free diet both the intestinal abnormality and the cutaneous manifestations of the disease are controlled, suggesting that a mucosal immune response is important in the pathogenesis of DH. Although patients with DH continue to ingest gluten only 40-50% have evidence of an ongoing mucosal immune response in their serum. In order to investigate directly the mucosal immune response in patients with DH the antibody response to dietary antigens was analyzed in intestinal secretions and compared to that found in the serum. Intestinal secretions from six patients with DH and five normal subjects were collected using an intestinal lavage solution and analyzed for total IgA, IgG, IgM, and IgA subclasses, and for IgG, IgA, and IgM antibodies against the dietary antigens bovine beta-lactoglobulin and gliadin. Intestinal secretions from patients with DH contained more IgA than those from normal subjects (mean total IgA: DH = 2.3 mg/ml; normal subjects (NL) = 0.143 mg/ml, P = 0.017). This increase in IgA in intestinal secretions from patients with DH was composed primarily of IgA1 (intestinal IgA: 86% IgA1, 14% IgA2; NL gut secretions: IgA1 = 54%; IgA2 = 46%). Increased IgA antibodies directed against beta-lactoglobulin and gliadin were detected in gut secretions of two of six patients with DH and in none of the normal subjects. Serum IgA antibodies against beta-lactoglobulin and gliadin were detected only in the two subjects who had detectable IgA antibodies in their intestinal secretions. Serum and intestinal IgA anti-beta-lactoglobulin antibodies had similar isoelectric spectrotypes (pI 5.0-6.5), IgA subclass composition, and antigenic reactivity by immunoblot analysis, demonstrating the close relationship between the serum and intestinal IgA antibodies. These data demonstrate that in patients with DH an ongoing mucosal immune response is present in the gut as evidenced by a significantly increased concentration of IgA, predominately IgA1. The strong correlation between detectable serum and intestinal IgA antibodies against dietary antigens demonstrates that the lack of serum IgA antibodies against dietary antigens in some patients with DH is not due to the presence of "blocking" IgA anti-dietary antigen antibodies in intestinal secretions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2000

How to develop globular proteins into adhesives.

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.

Adhesives↗
PubMed1994

Milk whey proteins in plasma of sows: variation with physiological state.

The whey proteins alpha-lactalbumin and beta-lactoglobulin have been investigated as potential markers of mammary development in sows by measuring their concentrations in plasma. The whey proteins were isolated from porcine milk by gel filtration, ion-exchange and hydrophobic interaction chromatography, characterized by several criteria and used to raise antibodies. Specific radioimmunoassays were set up for porcine alpha-lactalbumin and beta-lactoglobulin and validated for use in porcine blood and milk. Plasma levels of the whey proteins were measured in sows that were pregnant, suckling litters post partum, weaned abruptly at birth or were pregnant but mastectomized. Both whey proteins showed similar patterns in plasma post partum, falling from a maximum 1 d after parturition to values < 0.02% those in milk by day 4-5 post partum in suckling sows and showing a transient peak associated with early involution before declining to very low concentrations in non-suckling sows. alpha-Lactalbumin was first detected in the last week prepartum, rising markedly in the 3 d before parturition, correlated with rising prolactin (r = 0.986) and falling progesterone (r = -0.998). beta-Lactoglobulin rose much earlier from 5 weeks prepartum, at the time when lobulo-alveolar mammary development is occurring, and correlated (r = 0.929) with oestradiol-17 beta. In mastectomized sows, concentrations of whey proteins in plasma were reduced by 90% or more when compared with intact animals, though following a similar pattern. This study shows that whey protein concentrations in plasma vary with physiological state and reflect aspects of the development of the mammary gland. The very different profiles for alpha-lactalbumin and beta-lactoglobulin prepartum indicate that they are differently controlled.

Animals↗
PubMed2004

High pressure thermal denaturation kinetics of whey proteins.

Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (-2 to 70 degrees C), and the resulting degree of denaturation of beta-lactoglobulin A and B and alpha-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of beta-lactoglobulin and alpha-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both beta-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from -10 to about -30 ml mol(-1), which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol(-1) obtained for beta-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.

Animals↗
PubMed2006

Investigation of the lactosylation of whey proteins by liquid chromatography-mass spectrometry.

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.

Gas Chromatography-Mass Spectrometry↗
PubMed2005

Detergent binding as a sensor of hydrophobicity and polar interactions in the binding cavities of proteins.

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic detergents, in contrast to the cationic detergents, had no tendency to unfold the proteins at high concentrations.

Amino Acid Sequence↗
PubMed2003

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Alleles↗
PubMed2003

Avidity progression of dietary antibodies in healthy and coeliac children.

In most individuals minute amounts of food proteins pass undegraded across the intestinal mucosa and trigger antibody formation. Children with coeliac disease have enhanced antibody production against gliadin as well as other dietary antigens, e.g. beta-lactoglobulin, in cow's milk. Antibody avidity, i.e. the binding strength between antibody and antigen, often increases during antibody responses and may be related to the biological effectiveness of antibodies. The aim of the present study was to determine the avidity of serum IgG antibodies against beta-lactoglobulin and gliadin in healthy children during early childhood and compare these avidities to those found in children with coeliac disease. The average antibody avidity was analysed using a thiocyanate elution assay, whereas the antibody activity of the corresponding sera was assayed by ELISA. The avidity of serum IgG antibodies against beta-lactoglobulin as well as gliadin increased with age in healthy children, even in the face of falling antibody titres to the same antigens. Children with untreated coeliac disease had IgG anti-beta-lactoglobulin antibodies of significantly higher avidity than healthy children of the same age, and the same trend was observed for IgG antigliadin antibodies. The present data suggest that the avidities of antibodies against dietary antigens increase progressively during early childhood, and that this process seems to be accelerated during active coeliac disease.

Aging↗
PubMed1995

Stat5 as a target for regulation by extracellular matrix.

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine beta-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of beta-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with gamma-interferon activation site-related motifs within the beta-lactoglobulin promoter, which we show are required for matrix dependence of beta-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.

Animals↗
PubMed2000

Functional cavity dimensions of tear lipocalin.

PURPOSE: We calibrated the cavity of tear lipocalin with a series of fluorescent labeled lipids of increasing chain length and varying diameter. METHODS: Cavity length was assessed with competitive fluorescent assays in which DAUDA was displaced from apo-tear lipocalin with ligands of increasing carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC(50)) were compared. Functional diameters of tear lipocalin and beta-lactoglobulin were estimated with fatty acids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coordinates. RESULTS: In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 A) but remained constant from C18-C24. The cavity length of other lipocalins in crystal form were similar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to beta-lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor beta-lactoglobulin bound P646 in their respective cavities. The calculated inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16-22A. CONCLUSIONS: Tear lipocalin is more promiscuous than beta-lactoglobulin or retinol binding protein because of a greater functional diameter. Differences in ligand specificity of the various lipocalins can not be explained simply by variation in cavity length or the intersheet distances at the calyx mouths as determined by crystal structure. Other factors may influence ligand specificity such as size and/or dynamic motion of loops between the beta strands.

Carrier Proteins↗