Drug-binding and other physicochemical properties of a large tryptic and a large peptic fragment of human serum albumin.

The diazepam-binding behaviour of a large tryptic and a large peptic fragment of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate the primary diazepam-binding site on the albumin molecule. The analytical set-up of the FPLC was used to find the optimum experimental conditions for isolating the fragments. Conventional columns with a 100-fold higher loading capacity than the analytical FPLC columns were used to isolate large amounts of the fragments. The isolation procedure for the tryptic fragment (45 kDa, domains two and three of the albumin structure) is described in this paper. The description of the isolation procedure for the peptic fragment (46 kDa, domains one and two of the albumin structure) is published elsewhere (Bos, O.J.M., Fischer, M.J.E., Wilting, J. and Janssen, L.H.M. (1988) J. Chromatogr. 424, 13-21). The induced ellipticity of the diazepam-fragment complexes as well as the affinity of diazepam to the fragments turned out to be pH dependent. This pH dependence occurs in the region of the neutral to base transition of the albumin molecule. Difference CD-spectra of the proteins showed that the tryptic fragment and albumin have similar diazepam-binding properties, whereas the peptic fragment has different diazepam-binding properties. This result is in line with our equilibrium dialysis experiments which showed that the affinity of diazepam to the tryptic fragment and to albumin is of the same order of magnitude, whereas the affinity of diazepam to the peptic fragment is several orders of magnitude lower. On the basis of these results, it can be concluded that the tryptic fragment contains the primary diazepam-binding site and the peptic fragment one or more secondary diazepam-binding sites. This means that at least the main part of the primary diazepam-binding site is located in domain three of the albumin structure.

Mechanism by which warfarin binds to human serum albumin. Stopped-flow kinetic experiments with two large fragments of albumin.

In order to obtain information about the kinetics of the process by which warfarin binds to human serum albumin at a molecular level, we performed stopped-flow kinetic experiments on albumin and on a large peptic fragment (residues 1-387) and a large tryptic fragment (residues 198-585) of albumin. From these experiments it can be concluded that the first interaction between warfarin and the proteins is almost certainly diffusion-controlled and is dependent on the net charges of the reactants. The next step in the binding process involves the formation of an activated warfarin-protein complex, whereafter the final complex is formed. The warfarin-albumin complex forms more slowly than the warfarin-fragment complexes, because the formation is sterically hindered by the albumin structure. We think it very unlikely that albumin has an oblate ellipsoid structure; it is much more likely to have a U-shaped structure, where the domains make contact with each other. If this hypothesis is correct, then this indicates that the domains do not act independently of each other. The formation of the activated warfarin-albumin complex is further influenced by the conformational state of the albumin molecule, i.e. the N-B transition. The possible role of this N-B transition in albumin-mediated transport of drugs through cellular membranes is discussed.

Steroid binding to human serum albumin and fragments thereof. Role of protein conformation and fatty acid content.

The binding properties of the steroids testosterone and pregnenolone to human serum albumin (HSA) and derived fragments of albumin have been investigated by means of equilibrium dialysis and circular dichroism. The 46 kDa peptic fragment (P46) of HSA comprises domains one and two of the HSA structure, whereas the other fragment, the 45 kDa tryptic fragment (T45) is composed of domains two and three. A comparison of the binding behaviour of the steroid ligands to HSA and its fragments showed that the single primary testosterone binding site in all probability is located in the second domain of the HSA molecule. For pregnenolone it was found that at least two primary binding sites are present, also located in domain two. Both steroids show pH dependent binding profiles in the case of HSA and the P46 fragment. The binding of the steroids to the T45 fragment seems to be pH independent. The same phenomenon was observed with the circular dichroism experiments, indicating a link between the steroid binding properties and the structural behaviour of the proteins. In fact, the binding properties of the steroids can be assigned to the neutral-to-base (N-B) transition. The possible role of fatty acids as modulators in the transport processes of steroids in the body is discussed.

Location and characterization of the warfarin binding site of human serum albumin. A comparative study of two large fragments.

The warfarin binding behaviour of a large tryptic fragment (residues 198-585 which comprise domains two and three) and of a large peptic fragment (residues 1-387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate and characterize the primary warfarin binding site. The induced ellipticity of the warfarin-peptic fragment complex turned out to be pH dependent. This pH dependence occurs in the region of the neutral-to-base transition of the albumin molecule. The induced ellipticity of the warfarin-tryptic fragment complex is pH independent. Difference CD-spectra showed that the peptic fragment and albumin have similar warfarin binding properties whereas the tryptic fragment has deviant warfarin binding properties. The equilibrium dialysis experiments showed that the affinity of warfarin to the peptic fragment and to albumin is practically the same whereas the affinity of warfarin to the tryptic fragment is a factor 2-8 lower than the affinity of warfarin to albumin. Our results indicate that the main part of the primary warfarin binding site is located in domain two of the albumin structure and that domain one plays an important role in the N-B transition of albumin.

Location and characterization of the suramin binding sites of human serum albumin.

The objective of the present study was to investigate the location of the high-affinity suramin binding sites on the human serum albumin molecule. For this purpose, circular dichroism and equilibrium dialysis experiments were performed on the interaction between suramin and a large peptic and a large tryptic fragment of albumin, the former comprising domains one and two of the albumin structure and the latter domains two and three. The equilibrium dialysis experiments revealed that albumin and the fragments have a comparable total affinity for suramin. Furthermore, all three proteins display a similar pH dependence of the unbound fraction of suramin. The circular dichroism experiments revealed that only the suramin-albumin and the suramin-peptic fragment complexes can undergo the pH dependent neutral-to-base or N-B conformational change, whereas the suramin-tryptic fragment complex lacks this ability. It is likely that the main parts of the high-affinity binding sites for suramin are located in domain two of the albumin molecule. The nature of these binding sites is discussed. The deprotonation of histidine and other positively charged residues taking part in salt bridges between suramin and albumin is, in all probability, the main cause of the decrease in affinity of suramin for albumin as the pH is raised from 6 to 9.

The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes.

The effects of purified albumin species and albumin fragments (0.2-1% w/v) on short-term (4 h) steroid secretion by immature rat Leydig cells, in the presence of a maximally stimulating dose of luteinizing hormone (LH), were investigated. Human albumin and the peptic fragment (comprising residues 1-387) enhanced pregnenolone production in isolated rat Leydig cells, whereas chicken albumin and the tryptic fragment (comprising residues 198-585) were not active. This stimulatory effect of human albumin and the peptic fragment correlated with the potential of these proteins to undergo a pH-dependent neutral-to-base transition as measured by circular dichroism. The tryptic fragment and chicken albumin did not have the potential to undergo such a transition. The pH-dependent conformational changes of albumin and fragments thereof occurred in parallel with a change in the binding affinity for testosterone and pregnenolone. The fatty acid oleic acid and the drug suramin, only when present in a molar ligand-to-albumin ratio equal to or higher than 2, inhibited the albumin-mediated stimulation of steroid production. These data show that the stimulatory effects of albumin species on LH-induced Leydig cell pregnenolone production depend on their fatty acid content and correlate with the potential of these molecules to undergo conformational changes. It is unknown via which mechanisms albumin exerts its stimulatory effect, but the LH action through the cyclic AMP pathway seems not to be affected.

The uptake of the trypanocidal drug suramin in combination with low-density lipoproteins by Trypanosoma brucei and its possible mode of action.

In plasma, a significant part of suramin circulates in tight association with low-density lipoproteins (LDL). At therapeutically obtainable concentrations (100 microM) of suramin, about 85% of the total amount of the drug was bound to proteins, approximately 15% of which was bound to LDL. The molar ratio of suramin bound to LDL in serum was 7.5. The capacity of the high-affinity binding sites of LDL were 6.6 x 10(6) M-1, both in Tris buffer and in ultrafiltrate of serum. Suramin (100 microM) decreased the uptake of host LDL through receptor-mediated endocytosis by Trypanosoma brucei, with approximately 50%. LDL served as the only carrier for suramin uptake. Serum albumin, another important carrier for suramin in blood, was not able to promote suramin uptake, neither was delipidified plasma. The suramin taken up by T. brucei was recovered, in part, in the lysosomal fractions. It is suggested that deprivation of the parasite from cholesterol and phospholipids by an inhibition of the uptake of LDL, contributes to the mode of action of suramin, in addition to the many other effects that the drug may exert on the parasite. The toxic side-effects of suramin on the host are discussed in the light of its association with circulating lipoproteins.

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties.

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).

Procedure for the isolation of a large peptic fragment of human serum albumin.

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46,000. The fragment could be assigned to segment 1-387 and therefore consists of domains 1 and 2 of the albumin structure. A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin.

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.

Virus validation of pH 4-treated human immunoglobulin products produced by the Cohn fractionation process.

To assess the virus reducing capacity of Cohn's cold ethanol fractionation process for the production of intravenous (IVIg) and intramuscular (IMIg) immunoglobulin products, and treatment of these products at pH 4, a validation study of virus removal and/or inactivation was performed using both lipid-enveloped viruses [human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and pseudorabies virus (PSR)], and non-lipid-enveloped viruses [(simian virus 40 (SV40) and encephalomyocarditis virus (EMC)]. For the cold ethanol fractionation process, overall reduction factors of 3.0 logs, > or = 2.6 (< 5.5) logs, 4.6 logs, 5.8 logs and > or = 2.6 (< 6.2) logs were found for HIV, BVDV, PSR, SV40 and EMC, respectively. For all tested viruses the precipitation of fraction III from fraction II + III was the most effective step. From the overall reduction factors it appears that cold ethanol fractionation, although capable of reducing viral infectivity to a significant extent, is not sufficient to meet the requirements of regulatory bodies for viral safety of immunoglobulin products. However, pH 4 treatment contributes effectively to the viral safety of the final products. Treatment at pH 4.05 and 37 degrees C for 16 h, as is applied to IVIg, yields reduction factors of > or = 8.4 logs, > or = 4.0 logs, > or = 7.1 logs, 4.8 logs and 1.4 logs for HIV, BVDV, PSR, SV40 and EMC, respectively. The effectiveness of this process step could be enhanced by extending incubation to 40 h at pH 4.25 compared to 16 h at pH 4.05. The extended incubation, as applied in the production of IMIg, yields a reduction of infectivity of SV40 by > or = 5.5 (< 8.0) logs and of EMC by > or = 4.1 (< 7.1) logs. Storage of IMIg, which is formulated as a solution, at 2-8 degrees C also contributes to virus safety. For storage periods of 8 weeks or longer, reduction factors of 2 to 6 logs were found for all viruses, except for BVDV which remained unaffected. These data indicate that the production processes for IVIg and IMIg as described here have sufficient virus reducing capacity to achieve a high margin of virus safety.

Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues.

Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes.