The glycan structure of albumin Redhill, a glycosylated variant of human serum albumin.

Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (-1 Arg, 320 Ala-->Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.

Effect of oxidative stress on the structure and function of human serum albumin.

PURPOSE: Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esterase-like properties were studied. METHODS: Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p-nitrophenyl acetate as a substrate. RESULTS: Far-UV and near-UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO-HSA and CT-HSA but not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross-linking and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO-HSA and CT-HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT-HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level. CONCLUSIONS: Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase-like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin.

PURPOSE: The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. METHODS: Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. RESULTS: The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: "competitive-like" strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly "independent". CONCLUSIONS: The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.

Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.

In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time.

PURPOSE: Recombinant human serum albumin (rHSA), secreted by a Pichia pastoris expression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. METHODS: Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. RESULTS: Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. CONCLUSION: The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411.

Circular dichroism simulation shows a site-II-to-site-I displacement of human serum albumin-bound diclofenac by ibuprofen.

PURPOSE: The purpose of this study was to confirm the hypothesis that a site-II-to-site-I displacement takes place when some nonsteroidal anti-inflammatory drugs are displaced by another drug from their high-affinity binding site to a site of lower affinity on human serum albumin (HSA). METHODS: Diclofenac, sodium salt, was used as a representative example because of its prominent reversal of the Cotton effect. Effects of site-specific drugs on the free fraction of diclofenac were determined by equilibrium dialysis, and effects on induced circular dichroism (CD) of diclofenac bound to HSA were studied by CD and CD simulation techniques. RESULTS: Ibuprofen, a site-II-specific drug, altered the CD spectrum of the diclofenac-HSA complex at a molar ratio of 0.5 : 1 to that obtained at a higher ratio (5:1) without ibuprofen. The induced CD spectrum obtained in the presence of ibuprofen was very similar to one that assumed that all diclofenac displaced from its high-affinity binding site (site II) became rebound to a lower-affinity site (site I). The rebinding could be influenced by a free energy linkage between the two sites which would make site I (or parts thereof) more suitable for diclofenac binding. CONCLUSION: We have confirmed the existence of a site II-to-site I displacement, which is very striking and pharmacologically important, because the concentration of unbound drug being displaced is much lower than expected for a competitive mechanism.

Genetic variants showing apparent hot-spots in the human serum albumin gene.

The molecular defects of three different slow-migrating genetic variants of human serum albumin, albumins Kamloops (formerly RIH), Stirling and Amsterdam, previously characterized only by electrophoretic and dye-binding studies, are now reported. Two of them are proalbumin variants: sequential analysis of the purified whole proteins has established the mutation responsible for albumin Kamloops as -1Arg-->Gln, and for albumin Stirling as -2Arg-->His. A Glu-->Lys substitution in position 570 of the mature albumin molecule was determined in albumin Amsterdam by sequential analysis of two abnormal tryptic fragments. The three alloalbumins are caused by single-base changes all of which seem to represent hot-spots in the albumin gene. The possible functional consequences of the presence of a circulating alloalbumin are discussed.

Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition.

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition.

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin.

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.

Albumin genetic variability in South America: Population distribution and molecular studies.

A total of 5,020 individuals living in two southern Brazilian states were screened in relation to albumin types; two variants were found, in Passo Fundo (Nagasaki 2) and Vera Cruz (Tradate 2). Another variant, detected in the northeast, was identified as Porto Alegre 2, which also occurs in other places in Brazil, as well as in India, Pakistan, and Turkey. The results were integrated with those obtained in other studies in South America, yielding a total of 16,941 Amerindians and 23,839 non-Indian subjects. Molecular and physiological studies performed in some of the variants suggested clues to explain the restricted distribution of albumin Yanomama 2 and the widespread occurrence of albumin Maku. Am. J. Hum. Biol. 11:359-366, 1999. Copyright 1999 Wiley-Liss, Inc.

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.

Construction and use of a detergent-sensitive electrode to measure dodecyl sulfate activity and binding.

We describe the construction and use of a dodecyl sulfate-sensitive electrode cell to measure the activity of the detergent in biological samples. The electrode is based on the incorporation of a cetyltrimethylammonium/dodecyl sulfate complex in a siloxane polymer membrane. The cell records changes in the activity of SDS from 10(-6) to 10(-5) M SDS up to the critical micellar concentration. In aqueous solutions the cell follows Nernst' law with an electrometric response which is not affected by protein per se, but is modified by supporting electrolytes like NaCl. We demonstrate by comparison with equilibrium dialysis that the electrode can be used both to detect the high-affinity binding sites of serum albumin for SDS and to follow cooperative binding of the detergent to serum albumin, ovalbumin, and beta-lactoglobulin in the concentration interval 10(-4)-10(-3) M of unbound SDS. We conclude that the electrode has properties which should enable its use to monitor changes in SDS activity during interaction with biological material. The electrode may also be used to measure the activity of other detergents which, like SDS, form a sparingly soluble complex with cetyltrimethylammonium bromide.

Genetic variation in human serum albumin: a 313 Lys-->Asn mutation in albumin reading identified by PCR analysis.

An early case of bisalbuminaemia was reported in this journal in 1964, with the name Albumin Reading added later. Its use in electrophoretic comparisons led to some new variants being described as 'of the Reading type' on this basis alone. Protein sequencing and DNA studies have since found the single point mutation 313 Lys-->Asn common to this type, but the eponymous variant has not, until recently, been available for study. We now report on its characterisation using PCR analysis with allele-specific oligonucleotide primers, a method also applicable to studies of the population distribution of variants. We also draw attention to the need to link clinically-significant effects to individual variants of known structure.

Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA.

Three new genetic variants of human serum albumin have been detected in Italy by routine clinical electrophoresis. Albumin Milano Slow is common in Northern Italy, while albumins Liprizzi and Trieste, which are fast migrating, are rare and local variants. Isoelectric focusing analysis of the CNBr fragments obtained from the carboxymethylated alloalbumins in all cases localized the mutation to fragment CB5 (residues 330-446). The modified CNBr fragments were isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptides revealed that all the variants are caused by single point mutations: Trieste, Lys359-->Asn, Milano Slow, Asp375-->His, and Liprizzi, Arg410-->Cys. These results were confirmed by sequence determination of a variant V8 peptide in the case of Trieste, and by DNA sequence analysis for the other two variants. The DNA analysis showed a G-->C transversion at nucleotide position 11969 for albumin Milano Slow, and a C-->T transition at position 13251 for Liprizzi. The latter represents a mutation at a hypermutable CpG dinucleotide site. Albumins Trieste and Milano Slow, as most of the variants thus far described, have mutations involving residues on the surface of the molecule. In contrast, albumin Liprizzi represents the first example of a mutation in the most active binding pocket of the molecule, placed in subdomain IIIA.

Effect of genetic variation on the fatty acid-binding properties of human serum albumin and proalbumin.

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.

Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy.

A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.