Structural characterization of a glycoprotein variant of human serum albumin: albumin Casebrook (494Asp → Asn)

Albumin Casebrook is an electrophoretically slow genetic variant of human albumin with a relative molecular mass 2.5 kDa higher than normal albumin. It constitutes about 35% of total serum albumin in heterozygous carriers. The decrease in negative charge observed on incubation with sialidase suggested the presence of a carbohydrate moiety and the normalization of molecular weight following treatment with Endo-F indicated that this was an N-linked oligosaccharide. Partial acid hydrolysis and limited tryptic digestion established that the oligosaccharide was located in the C-terminal domaine, between residues 367 and 585. Tryptic, chymotryptic and S. aureus V8 proteinase digestions were carried out and the resulting glycopeptides were purified on concanavalin A-Sepharose. Peptide mapping of bound and unbound fractions followed by amino acid composition and sequence analysis, established a point mutation of 494 Asp → Asn. This introduces an Asn-Glu-Thr N-linkrf oligosaccharide attachment sequence centered on Asn-494 and explains the increase in molecular mass. There was no apparent pathology associated with the presence of this new glycosylated albumin, which was detected in two unrelated individuals of Anglo-Saxon descent.     

Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer

A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens.     

Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using (19)F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.     

Adducts of glycosylated serum albumin with amino acids

Glycosylation of human serum albumin was conducted by its long incubation with the excess either of D-glucose or D-glucose-6-phosphate at 37 degrees C. The glycosylated fractions were isolated by the cation-exchange chromatography on CM-cellulose. The quantity of glucose bound covalently with protein was determined by thiobarbituric acid. The glucose-modified human serum albumin forms stable adducts with amino acids. These complexes are, evidently, produced as a result of the Schiff's base formation between the carbonyl group of the ketoamine adduct of glucose with protein and primary amino group of amino acid further followed by the Amadori rearrangement.     

The fatty-acid-induced conformational states of human serum albumin investigated by means of multiple co-binding of protons and oleic acid.

The binding of oleic acid to human serum albumin causes progressive changes in (a) the pK of some amino acid residues, as detected by pH-stat titration and (b) the induced molar ellipticities of albumin-bound drugs (diazepam and oxyphenbutazone), as measured by c.d. It is concluded that albumin undergoes several conformational transitions as the amount of oleic acid bound increases from 0 to about 9 molecules/molecule of protein. At least three different conformations of the protein seem to be involved. These conformations can be linked with the three classes of oleic acid-binding sites on albumin.     

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.     

Use of amphotericin B as optical probe to study conformational changes and thermodynamic stability in human serum albumin

The binding of polyene antibiotic amphotericin B to serum albumin was studied using absorption, fluorescence, and circular dichroism techniques. A hypochromic effect was observed in the absorption spectrum of amphotericin B in the presence of albumin with maxima at 366 nm, 385 nm, and 408 nm, which correspond to the absorption of the monomeric form of amphotericin B. A modification on the circular dichroism spectrum of amphotericin B in the presence of albumin was observed at bands 329 nm and 351 nm (excitronic interaction), which suggests that only amphotericin B monomer is bound to the protein. Amphotericin B perturbs the specific markers for sites I, II, and fatty acid binding site bound to these sites, suggesting that amphotericin B interacts with a great binding area in albumin. Lysines 199 and 525 in albumin participate in the molecular interaction between amphotericin B and the protein. The absorption spectrum of amphotericin B bound to albumin was sensitive to the chemical and thermal treatment of the protein, to neutral-basic transition of albumin and to conformational changes induced by the binding of other ligands to this protein.     

Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein.

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary 'complex-type' with terminal fucose; IIA, biantennary 'complex-type' without fucose; IIB, biantennary 'complex-type' with fucose; III, a mixture of 'high-mannose' chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of 'high-mannose' type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with 'complex-type' chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three 'complex-type' chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.     

Effect of human serum albumin on drug metabolism: structural evidence of esterase activity of human serum albumin

Human serum albumin (HSA) is the most abundant plasma protein in the human body with a plasma concentration of 0.6mM. HSA plays an important role in drug transport and metabolism. Enzymatic activity of HSA on different substrates or drugs has been studied and documented. The structural mechanism of this activity, however, is unknown. In this study, we have determined the crystal structures of HSA-myristate in a complex of aspirin and of salicylic acid, respectively. The crystal structure of HSA-myristate-aspirin illustrates that aspirin transfers acetyl group to Lys199 and is hydrolyzed into salicylic acid by HSA. The hydrolysis product, salicylic acid, remains bound to HSA at a similar location, but it shows a very different orientation when compared with the salicylic acid in the HSA-myristate-salicylic acid ternary complex. These results not only provide the structural evidence of esterase activity of HSA, and demonstrate the conformational plasticity of HSA on drug binding, but also may provide structural information for the modulation of HSA-drug interaction by computational approach based on HSA-drug structure.     

Determination of binding characteristics as a measure for effective albumin using different methods

Background & aimsTransport functions of albumin are of clinical and pharmacological interest and are determined by albumin's properties like posttranslational modifications or bound ligands. Both are affected in pathological conditions and in therapeutic grade albumin solutions. The term effective albumin concentration was introduced as a measure of functionally intact albumin. Our aim was to evaluate the impact of ligands and modifications with different approaches as a measure of effective albumin.Approach & resultsWe used a spin labelled fatty acid and dansylsarcosine to characterize binding properties of albumin i) prepared from plasma of patients and healthy control donors, ii) measured directly out of plasma, iii) research grade albumin, iv) in vitro modified albumin, and v) therapeutic infusion solutions before and after removal of stabilizers.Bilirubin is the main determinant for binding function in patients' albumin. In in vitro prepared albumin bound fatty acids correlated with impaired binding. Human nonmercaptalbumin1, not human nonmercaptalbumin2, showed reduced binding properties. Binding and transport function of therapeutic albumin was severely impaired and restored by filtration. Glycation of research grade albumin had no effect on the binding of dansylsarcosine and only a minor effect on fatty acid binding.ConclusionsOur results suggest that effective albumin -in terms of binding properties- is primarily determined by bound ligands and only to a minor extent by posttranslational modifications. Characterizing albumin directly from plasma better reflects the physiological situation whereas in the case of therapeutic grade albumin stabilizers should be removed to make its binding properties accessible.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Glycosylation of endogenous protein(s) of the rough and smooth microsomes by a lipid sugar intermediate

Summary Several problems regarding the protein acceptor of the oligosaccharide from GEA (glucosylated endogenous acceptor) were investigated in the present work using rat liver microsomal subfractions. It was found that the acceptor molecule is present in rough and smooth liver microsomes. Furthermore both fractions have closely similar specific activities. The problem of whether nascent peptides must be ribosome bound for glycosylation to occur was studied. The results suggests that binding of peptides to ribosomes is not a necessary condition for the transfer of GEA oligosaccharide to protein. The increase in specific activity found after partial release of the microsomal vesicular content suggests that the acceptor protein for GEA is membrane bound. Evidence obtained in attempting to elucidate whether nascent or completed chains are glycosylated favours the later possibility.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.     

The binding of flavopiridol to blood serum albumin

Flavopiridol is a potent cyclin-dependant kinase (CDK) inhibitor and is in clinical trials for anticancer treatment. A limiting factor in its drug development has been the high dosage required in human clinical trials. The high dosage is suggested to be necessary because of significant flavopiridol binding to human blood serum. Albumin is the major protein component of blood serum and has been suggested as a likely high affinity binding target. We characterized the binding of human serum albumin to flavopiridol using circular dichroism (hereafter CD). Flavopiridol bound to human serum albumin has a diagnostic CD binding peak at 284 nm. The diagnostic CD binding peak was unobservable for flavopiridol with bovine serum albumin, using the same experimental conditions. However, under higher albumin concentrations a small CD signal is observed confirming, flavopiridol binds to bovine serum albumin as well.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

Characterization of spin-labelled fatty acids and hematoporphyrin binding sites interactions in serum albumin

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).     

The Antarctic toothfish (Dissostichus mawsoni) lacks plasma albumin and utilises high density lipoprotein as its major palmitate binding protein

Plasma from the Antarctic toothfish, Dissostichus mawsoni, a member of the advanced teleost Nototheniidae family, was analysed. Agarose gel electrophoresis showed a major diffuse anionic protein that bound [14C]palmitic acid but not 63Ni2+, and two more cationic proteins that bound 63Ni2+ but not palmitate. Oil Red O staining following cellulose acetate electrophoresis indicated that the palmitate binding protein was a lipoprotein. Two-dimensional electrophoresis showed that this palmitate binding band was composed of three proteins with M(r) of 11, 30, and 42 kDa, without any trace of material at approximately 65 kDa, the mass of albumin. N-terminal sequencing of the palmitate binding band gave a major sequence of DAAQPSQELR-, indicating a high degree of homology to apolipoprotein A-I (apo-AI), the major apolipoprotein of high density lipoprotein (HDL). N-terminal sequencing of the major nickel binding band produced a sequence with no homology to albumin. When ultracentrifugation was used to isolate the lipoproteins from Antarctic toothfish plasma, the palmitate binding protein was found solely in the lipoprotein fraction. In competitive binding experiments, added human albumin did not prevent palmitate binding to toothfish HDL. In conclusion, there is no evidence for albumin in Antarctic toothfish plasma and HDL assumes the role of fatty acid transport.     

The use of 8-anilino-1-naphthalenesulfonic acid as a reporter group molecule for circular dichroism and fluorescence measurements. The effect of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin.

The fluorescence probe ANS(8-anilino-1-naphthalenesulfonic acid) was employed as a reporter group molecule for circular dichroism and fluorescence measurements in order to investigate the effects of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin. Stearate as well as dodecylsulfate displaces ANS from the binding to both albumins. Besides this displacement, stearate and dodecylsulfate influence the fluorescence properties and the extrinsic Cotton effects on ANS bound to both albumins. It is suggested that the origin of these effects is a microdisorganization of the albumin structure, provoked by the binding of stearate and sodium dodecylsulfate. Each of the four extrinsic CD bands of bound ANS was influenced in a different manner by the addition of stearate and dodecylsulfate. Using the data of the fluorescence measurements and of the circular dichroism measurements it was possible to differentiate the effects of one ligand on both albumins and of both ligands on one albumin more efficiently than would have been possible using one of the two methods alone. It is suggested that the use of ANS as a reporter group molecule for fluorescence and circular dichroism measurements is a very good tool to detect small changes in the environment of ligand binding sites on protein molecules.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.