A stereochemical investigation of phosphoryl transfer catalysed by phosphoglucomutase by the use of alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate.

Fluorescence spectra of protoporphyrin bound to its most affinitive site on human serum albumin, bound to human haemopexin and dissolved in human plasma reveal that, when present in plasma, at least 90% of this porphyrin is bound to albumin. Human serum albumin binds protoporphyrin with an affinity KA = 3 X 10(9)M-1 in phosphate-buffered saline. The affinity of haemopexin for protoporphyrin is 4 times smaller. From these data it is concluded that less than 1% of plasma protoporphyrin is bound to haemopexin. Implications of the data for protoporphyrin transport and clearance are discussed.     

Role of apolipoprotein D in the transport of bilirubin in plasma

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.     

Conformational alteration in serum albumin as a carrier for pyridoxal phosphate: a distinction from pyridoxal phosphate-dependent glutamate decarboxylase

The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyridoxal-P) carrier, was investigated by using uv/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. Upon interacting with pyridoxal-P, the uv/visible absorption spectrum of BSA exhibits peaks at 330 and 392 nm due to the formation of a Schiff base. Pyridoxal-P quenches the fluorescence emission intensity (excited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polarization of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational change appears to be small since circular dichroism showed only a 2-4% decrease in the alpha-helical content of BSA and no change in the beta-sheet content, and differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, an antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitive sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxal-P, the results were compared with those for glutamate decarboxylase (GAD), a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofactor for activity. Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>10(6) M-1), distinct conformational alterations occur between the two proteins upon interacting with pyridoxal-P: a small conformational change for BSA versus a large conformational change for GAD. In contrast to the case of BSA, AET causes significant effects on both the uv/visible spectrum and fluorescence emission intensity of GAD, because GAD contains sensitive sulfhydryl groups. Factors such as disulfide bond and active site sequence were discussed to understand BAS as a carrier for pyridoxal-P and a pyridoxal-P-independent protein.     

Cicletanine binding to human plasma proteins and erythrocytes, a particular HSA-drug interaction

The binding of cicletanine to human serum, isolated proteins and red blood cells was studied in vitro by equilibrium dialysis. Our results show this drug is highly bound to serum (97.3%) at therapeutic levels. No saturation to the binding sites was seen. Human serum albumin was shown to mainly responsible for this binding (93.5%) with a saturable process characterized by one binding site with a moderate affinity (K = 75800 M-1) and a non saturable process with a low total affinity (nK = 6400 M-1). Like many basic lipophilic drugs, cicletanine showed a saturable binding to alpha-1-acid glycoprotein with one site and a moderate affinity (K = 38,800 M-1). Its binding to lipoproteins and red blood cells was weak and non saturable. Over the range of therapeutic concentrations, the unbound fraction in blood remains constant (3.6%). Moreover, interactions were studied using bilirubin and non esterified fatty acids at pathological concentrations and these endogenous compounds did not alter cicletanine binding human serum or to human serum albumin likewise cicletanine shared the diazepam-site on HSA but no inhibition could take place between cicletanine and the drugs sharing the same binding site in serum at therapeutic levels.     

Haem degradation in human haemoglobin in vitro. Separation of the contribution of the alpha- and beta-subunits.

As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found.     

Various enzymatic and nonenzymatic pathways of acetaldehyde transformation

In blood acetaldehyde is bound to hemoglobin and serum albumin and interacts with low-molecular-weight components--cysteine, glutathione. The association constants for human serum albumin and human hemoglobin are about 30 M-1 and 410 M-1, respectively. The SH-groups of hemoglobin play an essential role in the interaction of protein with acetaldehyde. The reversible complexes of acetaldehyde with proteins can transform into stable adducts after long incubation with albumin, some other blood proteins but not with immunoglobulins. Acetaldehyde participates as a substrate in the reaction of acetoin formation in homogenates of different tissues. The rate of acetoin formation is about 15 +/- 3 mmol/min per 1 gram of the tissue and essentially increases in the presence of pyruvate.     

The binding of silica to proteins from plasma and lungs of rat: in vitro

Silica dissolving out from the slate dust was found to bind with plasma protein and purified bovine serum albumin. At 24 h of incubation at 37 degrees C binding affinity of silica (microgram of silica bound/mg of protein) with plasma protein and bovine serum albumin was found to be 0.59 and 0.44, respectively. By molecular exclusion chromatography using Sephadex G-200, silica binding protein of plasma was determined to be of mol. wt. around 67000. Similar proteins having silica binding capacity (mol. wt. 70000 and 85000) were also found in rat lung but these proteins unlike their plasma counterpart were glycoprotein in nature. Polyacrylamide gel electrophoresis of plasma and protein rich lung fraction show that proteins upon binding with silica undergo mobility changes. Significance of the existence of silica binding protein in plasma and lung of rat in relation to silica toxicity is discussed.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

Amyloid-beta peptides interact with plasma proteins and erythrocytes: implications for their quantitation in plasma

Amyloid beta peptides are bound rapidly in the plasma complicating an accurate assessment of their in vivo abundance by immunoassay procedures. The extent of Abeta immunoassay interference was used to estimate the Abeta binding capacity of purified plasma proteins, erythrocytes and whole plasma. Human serum albumin bound Abeta peptides rapidly with a 1:1 stoichiometry and at physiological concentrations was capable of binding over 95% of an input of 5 ng/ml Abeta. Purified alpha2-macroglobulin was able to bind Abeta peptides and at physiological concentration bound 73% of 5 ng/ml of Abeta. Erythrocytes also sequestered the Abeta peptides, showing a preference for binding Abeta 1-42. Incubation of 5 ng/ml of Abeta in plasma revealed that about 30% of the peptides were still detectable by immunoassay, presumably reflecting the binding of Abeta peptides with albumin and other plasma molecules. Thus, our studies reveal that both the soluble and formed elements of the blood are capable of sequestering Abeta peptides. To avoid underestimating plasma Abeta values, we employed an improved column chromatography method under denaturing conditions to liberate Abeta from its associations with plasma proteins. Quantification of Abeta 40 and 42 levels in plasma from both normal and AD individuals after chromatography showed a large overlap between AD and control groups, despite the very large pool of Abeta present in the AD brains. The potential origins of the plasma Abeta pool are discussed.     

The binding of beta-2-microglobulin to renal brush-border membrane: affinity measurement, inhibition by serum albumin

In the kidney, filtered proteins are rapidly reabsorbed so that the final excretion is less than 0.1% of the filtered amount for low molecular weight proteins such as beta 2-microglobulin and a few percent for albumin. In order to investigate the affinity of proteins for luminal membranes, rat renal brush-border membranes were incubated with 125I-labelled human beta 2-microglobulin and the initial binding rate determined by the filtration method. Scatchard plot analysis of binding rate revealed two types of binding sites: one with Km = 0.25.10(-6) M and Vmax = 0.1 nmol/min per mg protein and another with Km = 1.10(-5) M and Vmax = 1.3 nmol/min per mg protein. The lower affinity type is likely to represent non-specific binding the physiological role of which is to be discussed. The higher affinity sites seem to play the major role in binding rate. beta 2-Microglobulin initial binding is reversible, and inhibited by bovine serum albumin. Comparison of the time course of bound beta 2-microglobulin removal by unlabelled beta 2-microglobulin and by albumin suggests that these two proteins have a different internalization mechanism.     

Binding of norgestrel to human plasma proteins.

Binding of [14, 15-3H](+/-)-norgestrel to human plasma proteins has been investigated. Norgestrel showed greater affinity to plasma than to human serum albumin indicating specific norgestrel binding protein(s) in the plasma. alpha1-acid glycoprotein showed high affinity for norgestrel when compared with human serum albumin. The binding protein was eluted at pH 5.8 by step by step elution on a DEAE-cellulose column. Norgestrel binding to plasma proteins was not affected at 60 degrees C. The optimal binding occurred between pH 7 and 8. Ligand specificity of the binding protein revealed that progesterone was able to compete for the norgestrel binding sites, whereas corticosterone, testosterone, oestradiol, and norethindrone acetate did not show much competition. The molecular weight of the binding protein was found to be approximately 43 000. Sucrose density gradient analysis indicated that norgestrel bound to a macromolecular component of sedimentation coefficient 2.9 S. The association constant (Kass) and dissociation constant (Kdiss) of norgestrel-binding plasma protein was found to be 1.4-10(6) M-1 and 0.7-10(-6) M respectively. The number of binding sites was 0.5-10(-9) mol/mg protein. Norgestrel-binding protein in the plasma appeared to be a protein different from human serum albumin, corticosteroid-binding globulin and sex-steroid-binding protein. This binding protein showed some similarities to alpha1-acid glycoprotein.     

Binding of estrogen-3-sulfates to stallion plasma and equine serum albumin.

The binding of estrone-3-sulfate (E1-3-S) and estradiol-3-sulfate (E2-3-S) to adult stallion plasma was determined and compared with the binding to equine serum albumin (ESA). On the ESA molecule, two binding sites for E1-3-S with an association constant of 1.3 x 10(5) M-1 and several sites of weaker affinity were found; the data for E2-3-S showed the existence of four binding sites of moderate affinity (1 x 10(5) M-1) and several sites of weaker affinity. The removal of albumin from the stallion plasma resulted in the absence of binding of E1-3-S or E2-3-S, whereas the removal of glycoproteins resulted in binding parameters similar to those obtained with whole plasma. These results indicate that ESA is the only estrogen sulfate binder in horse plasma. Under physiological conditions, 95% of E1-3-S was bound to ESA.     

A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.     

Corticosteroid binding in plasma of Xenopus laevis. Modifications during metamorphosis and growth

The binding of corticosteroids has been studied by equilibrium dialysis at 4 degrees C and electrophoresis on polyacrylamide gel. Aldosterone was not bound specifically. Large amounts of corticosterone (B) were bound. A high affinity (Ka = 2.8 X 10(8) M-1) low capacity (70.1 nM) component was found in tadpoles. Its affinity and its electrophoretic mobility were not significantly modified during metamorphosis and growth until adult. It differs from mammalian CBG with respect to affinity, electrophoretic mobility and specificity. During growth, the capacity of the high affinity component increased significantly (173 nM in adults) and a low affinity (Ka = 2.0 X 10(5) M-1) high capacity (18.26 microM) component was detected. This last component has the same electrophoretic mobility as bovine serum albumin. These modifications can be related to the large increase in the level of plasma proteins (especially albumins). The concentrations of bound and free B deduced from our data indicate that in tadpoles the increase in the total concentration in the climax is not a good reflection of modifications of these two fractions since the change of free B is larger than that of bound B. This information is an important consideration in interpreting the physiological role of interrenal secretion during metamorphosis.     

The comparison of biophysical properties of DB-67 and its ester DB-67-4ABTFA determined by fluorescence spectroscopy methods

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

Effect of quinolinate on aminotransferases

Quinolinate inhibits several aminotransferases (ornithine, alanine, and aspartate). However, it is considerably more potent as an inhibitor of liver and heart cytoplasmic aspartate aminotransferase. It is a much less potent inhibitor of mitochondrial aspartate aminotransferases. Quinolinate is bound to the active site of cytoplasmic aspartate aminotransferase. It has a much greater affinity for the pyridoximine-P than the pyridoxal-P form of the enzyme. According to kinetic results, the inhibition or dissociation constant of quinolinate is 0.2 and 20 mm, respectively, for the pyridoxamine-P and the pyridoxal-P forms of the enzyme. Since quinolinate is mainly bound to the pyridoxamine-P form: (a) it is a potent competitive inhibitor of α-ketoglutarate but has little effect when α-ketoglutarate is saturating even if the level of aspartate is low; (b) it decreases the effect of α-ketoglutarate on the absorption spectrum of the pyridoxamine-P form; and (c) it enhances the effect of glutamate on the absorption spectrum of the pyridoxal-P form. Quinolinate is also apparently bound to the apoenzyme since it inhibits reconstitution by either pyridoxamine-P or pyridoxal-P. Since quinolinate is a competitive inhibitor of α-ketoglutarate, it is possible that part of the inhibitory effect of quinolinate on hepatic gluconeogenesis could result from quinolinate inhibiting the conversion of aspartate to oxalacetate by the cytoplasmic aspartate aminotransferase. Quinolinate has no effect on either rat or bovine liver glutamate dehydrogenase or on kidney glutamate dehydrogenase.     

Flurorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.