Stabilization mechanism of prostacyclin by human serum: an approach by binding kinetics using a stable prostaglandin I2 analogue, iloprost

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.     

Exposure of latent prostaglandin binding sites in the rat epididymal adipocyte membrane and the effects of albumin, heating and alkylating agents

Brief incubation (1 min) of the adipocyte isolated membranes at 60 degrees C caused an increase in prostaglandin E2 binding, similar to that obtained with albumin. The increase in the membranal binding capacity after a short heating of the membranes was concomitant with a substantial decline in the ability of albumin to induce a further increase in the binding capacity of the treated membranes. Pretreatment of the isolated adipocyte membranes with 10 mM N-ethylmaleimide inhibited the enhancement of prostaglandin E2 binding in the presence of albumin, but did not affect the prostaglandin E2 binding in the absence of albumin. Identical treatment of the isolated membranes with glutathione-maleimide, an impermeable SH reagent with comparable alkylation reactivity, enhanced the binding of prostaglandin E2 in the absence of albumin and failed to inhibit the enhancement of prostaglandin E2 binding in its presence. In contrast to the effect of albumin on prostaglandin E2 binding to the isolated membranes, albumin failed to alter prostaglandin E2 specific binding to intact adipocytes.     

Interaction of prostaglandins with blood plasma proteins. I. Binding of prostaglandin E 2 to human plasma proteins and its effect on the physiological activity of prostaglandin E 2 in vitro and in vivo

The binding of (PGE2) prostaglandin E2 to human plasma proteins was investigated by DEAE-Sephadex column chromatography and acrylamide gel electrophoresis, and quantitatively assessed by equilibrium dialysis. PGE2 added to human plasma in vitro was found to become mainly bound to plasma albumin. This binding was also demonstrated by adding PGE2 to human serum albumin solutions. The binding of PGE2 to human serum albumin inhibits the contraction-producing effect of PGE2 on the isolated gerbil colon in vitro. The depressor effect of PGE2 on the rat blood pressure was used to assess the in vivo effect of PGE2 albumin interaction. The blood pressure lowering activities of free and albumin-bound PGE2 were found to be the same when administered either intravenously or intraarterially. The significance of these observations with regard to estimation of PG concentration in whole blood or plasma, and their possible effects on PG metabolism is discussed.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Fatty acid binding protein (FABP) modulates prostaglandin E binding to rat epididymal adipocyte membrane similarly to albumin

Albumin enhances prostaglandin E2 (PGE2) binding to isolated epididymal adipocyte membrane and also binds PGE2 with low affinity. On the other hand, S-100, ovalbumin and albumin-stearate failed to bind PGE2, as shown by ultrafiltration, and also failed to enhance PGE2 binding to the isolated adipocyte membranes. These results suggested that albumin enhances PGE2 binding possibly by serving as a carrier for the prostaglandin molecules. 3 mM warfarin or 1 mM phenylbutazone inhibited PGE2 binding to albumin by 70% and 95%, respectively, but both drugs failed to affect the enhancement of PGE2 binding to the isolated adipocyte membrane in the presence of albumin. These results exclude the possibility that PGE2 bound to albumin is more accessible to the prostaglandin receptor than free PGE2 in solution. Finally it is shown that fatty acid binding protein (FABP), a cytosolic protein which binds specifically PGE1 but not PGE2, enhances PGE1 and PGE2 binding to isolated adipocyte membranes similarly to albumin. The physiological implications of these findings are discussed.     

Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1-fetoprotein and albumin.

We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.     

Binding studies on aluminum(III)-albumin interaction

The present paper reports a quantitative investigation on the binding of aluminum to human serum albumin. Equilibrium dialysis and a general thermodynamic approach have been used to determine the binding parameters. Two aluminum binding sites have been identified on the albumin molecule, involving sites with single and double occupancy modes. The binding properties of these two distinct sites appear to undergo reciprocal influences, suggesting a possible interaction between the corresponding protein moieties. Both coordination modes proceed from weak interactions, as shown by the binding energies calculated.     

Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.     

The competition of serum amyloid protein and apoprotein E for binding with human serum albumin]

The relationship between the concentration of serum amyloid protein (SAP) isolated from human serum and the parameters of the protein elution during gel-filtration and alos with the efficiency of Ca(2+)-dependent SAP binding with sepharose 4B was studied. The dissociation of the SAP oligomeric form in solution and the interaction of the protein with human serum albumin with fully reduced S--S bridges due to the introduction of the additional hydrophobic surface was shown. Apoprotein E isolated from human plasma very-low-density lipoproteins replaced SAP in the complex with albumin.     

Isomerization of prostaglandin H2 into prostaglandin D2 in the presence of serum albumin.

The prostaglandin endoperoxide, prostaglandin H2, decomposes in aqueous media mainly into prostaglandin E2. This paper shows that in the presence of serum albumin from a number of species prostaglandin H2 decomposes mainly into prostaglandin D2, an isomer of prostaglandin E2. The effect on endoperoxide decomposition exerted by serum albumin may well have physiological significance since intace endoperoxides can be released from tissues and since the biological properties of prostaglandins E2 and D2 are quite different.     

Analysis and use of the serum albumin binding domains of streptococcal protein G

Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.     

Interaction of prostaglandins with blood plasma proteins. Comparative binding of prostaglandins A2, F2α and E2 to human plasma proteins

1. The binding of prostaglandin A(2) and prostaglandin F(2alpha) to human plasma proteins was investigated by DEAE-Sephadex chromatography and polyacrylamide-gel electrophoresis. Both prostaglandins, when added to human plasma in vitro, were found to become bound mainly to plasma albumin. 2. The extent of binding of prostaglandins added to human plasma in low to moderate concentrations was found to be approx. 88, 73 and 58% for prostaglandins A(2), E(2) and F(2alpha) respectively. The order of affinities for the binding of the three prostaglandins to albumin appear to be A(2)>E(2)>F(2alpha). 3. The apparent association constants for the binding of these prostaglandins to human serum albumin were estimated to be approx. 4.8x10(4), 2.4x10(4) and 0.9x10(4) litre/mol for prostaglandins A(2), E(2) and F(2alpha) respectively. The results are compared with previously reported association constants for the binding of long-chain fatty acids to both human and bovine albumins.     

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.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Enkephalin binding systems in human plasma II: Leu-enkephalin serum albumin interaction

Enkephalins are released into the bloodstream of mammals by the adrenal medulla. Once they are in the blood, these peptides undergo a fairly rapid hydrolysis by several plasma-contained enzymes. However, a fraction of the enkephalins present in the plasma are bound to the serum albumin, and the bound peptides are almost completely intact even after a long incubation in the presence of serum enzymes. Therefore, it seems possible that the interaction with serum albumin can maintain the functional integrity of the circulating enkephalins. Moreover, serum albumins are extremely well characterized proteins and, therefore, a suitable model for the study of protein-enkephalin interaction in general. The present work is a first step in the study of the mechanism of serum albumin-enkephalin interaction. Apparently, ionic parameters are important in the binding phenomenon. Furthermore, the serum albumin conformational status seems to be relevant in the binding. Finally, the binding is followed by a limited rearrangement of the protein molecule.     

Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain

The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Spectroscopic Studies of the Supramolecular Interactions Between Uracil and 5-Hydroxy-6-Methyluracil with Bovine Serum Albumin and its Bilirubin Complex

Using fluorescence and absorption spectroscopy the interaction of bovine serum albumin and its bilirubin complex with uracil and 5-hydroxy-6-methyluracil in phosphate buffer at pH 7.4 was investigated. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, the radius of the quenching sphere and etc.) were determined. The interaction between serum albumin and uracils is carried out by the static quenching of protein fluorescence and has predominantly hydrophobic character. Using synchronous fluorescence spectroscopy the influence of uracil and 5-hydroxy-6-methyluracil on the conformational changes of the protein molecule was studied. Uracils effectively binds to bilirubin-albumin complex compared to free protein, which is caused by the interaction with tetrapyrrolic pigment in macromolecular complex. Molecular docking calculations also being presented.     

Binding interaction of a biological photosensitizer with serum albumins: a biophysical study

A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.