Large fragments of human serum albumin.

Large fragments of human serum albumin were produced by treatment of the native protein with pepsin at pH3.5. Published sequences of human albumin [Behrens, Spiekerman & Brown (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591; Meloun, Moravek & Kostka (1975) FEBSLett.58, 134-137]were used to locate the fragments in the primary structure. The fragments support both the sequence and proposed disulphide-linkage pattern (Behrens et al., 1975). As the pH of a solution of albumin is lowered from pH4 to pH3.5, the protein undergoes a reversible conformational change known as the N-F transition. The distribution of large fragments of human albumin digested with pepsin in the above pH region was critically dependent on pH. It appeared that this distribution was dependent on the conformation of the protein at low pH, rather than the activity of pepsin. The yields of the large fragments produced by peptic digestion at different values of pH suggested that the C-terminal region of albumin unfolds or separates from the rest of the molecule during the N-F transition. The similarity of peptic fragments of human and bovine albumin produced under identical conditions supports the proposed similar tertiary structure of these molecules.     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.     

Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Conformational transitions of the three recombinant domains of human serum albumin depending on pH

Human serum albumin (HSA) is a protein of 66.5 kDa that is composed of three homologous domains, each of which displays specific structural and functional characteristics. HSA is known to undergo different pH-dependent structural transitions, the N-F and F-E transitions in the acid pH region and the N-B transition at slightly alkaline pH. In order to elucidate the structural behavior of the recombinant HSA domains as stand-alone proteins and to investigate the molecular and structural origins of the pH-induced conformational changes of the intact molecule, we have employed fluorescence and circular dichroic methods. Here we provide evidence that the loosening of the HSA structure in the N-F transition takes place primarily in HSA-DOM III and that HSA-DOM I undergoes a structural rearrangement with only minor changes in secondary structure, whereas HSA-DOM II transforms to a molten globule-like state as the pH is reduced. In the pH region of the N-B transition of HSA, HSA-DOM I and HSA-DOM II experience a tertiary structural isomerization, whereas with HSA-DOM III no alterations in tertiary structure are observed, as judged from near-UV CD and fluorescence measurements.     

Anomalous salting-out behaviour of cyanogen bromide fragments of bovine serum albumin

The solubilities of bovine serum albumin and its two cyanogen bromide fragments comprising domain I and II+III of the protein in ammonium sulphate solution were studied at different pH and temperature and the salting-out parameters K_s and β were determined for the three proteins. The values of K_s and β obtained for the intact albumin at different pH were atypical of other globular proteins and were explained in terms of N-F transition and pH induced unfolding of the protein. The salting-out behaviour of the two fragments was, however, found to be significantly different from that of their parent molecule. In contrast to bovine serum albumin, the aqueous solubilities of the two fragments were highly dependent on temperature. Similarly, pH dependence of β for the two fragments was also different since it acquired a minimum value at about pH 4.0 as against its monotonic decrease with pH observed in intact albumin below pH 5.0. Anomalous salting-out behaviour of the two cyanogen bromide fragments has been attributed to the possible conformational changes that might occur during the course of their preparation under relatively harsher chemical conditions.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Circular dichroic and fluorometric studies on the acid-induced isomerization of bovine plasma albumin--1 -anilino-8-naphthalenesulfonate complex

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin--1-anilino-8-naphthalenesulfonate complex, BPA-ANS1.0 complex (molar ratio of added ANS to BPA = 1.0) were studied by measuring fluorescence and induced CD spectra of ANS. Decrease in the reciprocal of fluorescence polarization, increase in fluorescence intensity and blue shift of fluorescence of ANS in BPA-ANS1.0 complex were correlated with the initial part of the N-F transition and/or the N-F1 transition. Induced CD spectra of ANS showed positive bands at 250-258 and 320-350 nm and one negative band at 280 nm. Most of changes (decreases) in -[theta]280 were also correlated with the initial part of the N-F transition and/or the N-F1 transition. Changes in fluorescence parameters and induced CD spectra of ANS (-[theta]280) might indicate the conformational changes around a strong ANS binding site in the N-terminal domain (Reed et al. (1975), Jonas & Weber (1970) and Brown & Shockley (1982].     

Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions.

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.     

Isolation,characterization and effect of acidic pH on the unfolding-refolding mechanism of serum albumin domains

Three fragments,viz., BSA-CNBr_1–183, BSA-CNBr_184–582, and BSA-T_377-582 representing domains I, II + III and III of bovine serum albumin have been isolated and purified. The physicochemical properties have been investigated and compared with their parent albumin molecule. The values of Stokes radii (nm) and intrinsic viscosities (ml/g) have been determined to be 2.36, 3.30; 3.43, 4.36; and 2.40, 3.13 for the fragments BSA-CNBr_1-183 BSA-CNBr_184-582 and BSA-T_377-582 respectively. The acid induced unfolding-refolding transitions of intact albumin and the fragment BSA-T_377-582 have been shown to occur in two steps while the fragments BSA-CNBr_1-183 and BSA-CNBr_184-582 underwent single step transitions. The formation of the acid denatured states of intact albumin, BSA-CNBr_1–183 and BSA-CNBr_184-582 was accompanied by an increase of about 86, 56 and 44% in the values of intrinsic viscosities respectively. Since all the transitions were reversible, the values of equilibrium constants,K _D, were calculated. The analysis of the dependence ofK _D on pH indicated that the first transition (N-X) of albumin was caused due to the uptake of about 3 protons by the native albumin. The intermediate state,X, is converted to acid unfolded state,D, by taking up another two protons. A comparision of the results on intact albumin with that of its fragments revealed that the second transition of the fragment BSA-T_377–582 and the two single step transitions of the fragment BSA-CNBr_1-183 and BSA-CNBr_184-582 were much closer to the second transition (X-D) of the intact albumin. The first transition of albumin has been attributed to its domain III represented by the fragment BSA-T_377-582.     

CD-resolved secondary structure of bovine plasma albumin in acid-induced isomerization

Bovine plasma albumin (BPA) showed the acid-induced two-step transition, the N-F transition and acid-expansion. Changes in fractions of alpha-helix (f alpha), beta-form (f beta) and unordered form (fR) in the acid-induced isomerization of BPA were studied using the method of Chen et al. (1972) with two constraints: sigma fi = 1, 0 less than or equal to fi less than or equal to 1. pH-profiles of f alpha and fR showed the two-step change, one corresponding to the N-F transition and the other to the acid-expansion in 0.10 M KCl and in 0.02 M NaClO4. pH-profile of f beta showed one-step change, correlating to the later part (lower pH side) of the N-F transition. The N-F transition might thus involve the helix leads to beta and helix leads to coil transitions.     

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.     

Urea-induced structural transformations in bovine serum albumin

Urea-induced unfolding of bovine serum albumin and one of its fragments containing domain II + III has been studied by difference spectral and fluorescence emission measurements. The unfolding-refolding curves of both the proteins showed the presence of at least one stable intermediate when the transition was monitored at 288 nm. The presence of the intermediate was not detectable at 293 nm where only tryptophan contributed towards the protein absorption. However, both the proteins did show the presence of intermediate when the denaturation was monitored fluorometrically. Since domain III of the albumin is devoid of tryptophan, it is concluded that the formation of intermediate in the unfolding-refolding transition of serum albumin involves (i) unfolding of domain III, (ii) minor structural transformations in domain II, and/or (iii) the separation of the sub-domains of domain III from each other.     

Studies on the structure and mechanism of action of glycoside hydrolases. I. Purification and study of some factors affecting the activity of Rhizopus arrhizus (1 leads to 3)-beta-D-glucanase

The extracellular (1→3)-β-D-glucanase [(1→3)-β-D-glucan glucanohydrolase, E.C. 3.2.1.6] produced by Rhizopus arrhizzus QM 1032 was purified 165-fold by chromatography. The purified enzyme is basic, has a molecular weight of ≈ 10,000, and is unstable in dilute solution but may be stabilized by addition of human serum albumin. The pH-activity profile for the enzyme in the presence of serum albumin shows a peak at about pH 3.5–3.7 and a shoulder at pH 4.5–4.6, whereas in the absence of serum albumin the optimum pH is at pH 4.5–4.6, indicating the presence of two enzymic species, designated “pH 3. 5 activity” and “pH 4.6 activity”. In the presence of albumin the enzyme activity is resistant to inactivation by a wide range of reagents. Ammonium molybdate is, however, a powerful inhibitor of ldpH 3.5 activity” although a much poorer inhibitor of “pH 4.6 activity”. The enzyme activity is stable during heating at pH 3.5 in the presence of human serum albumin. Thus, 94.5 and 88.5 % of “pH 3.5 activity” and “pH 4.6 activity”, respectively, remained after heat treatment for 30 min at 68°. The enzyme is, however, essentially inactive at this temperature, even in the presence of albumin. To account for this finding, a temperature-dependent conformational change is proposed. The enzyme activity is not stable during heating at pH 4.6 in the presence of serum albumin. K_m values for action on laminaran are 0.54 mg/ml (pH 3.5) and 0.27 mg/ml (pH 4.6). For lichenan the corresponding values are 3.33 and 2.38 mg/ml. The V_max for enzyme action on lichenan is 35–40% higher than for action on laminaran at both pH values. Possible relationships between the two forms of the enzyme are briefly considered.     

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.     

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).     

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Changes in the electric dipole vector of human serum albumin due to complexing with fatty acids.

The magnitude of the electric dipole vector of human serum albumin, as measured by the dielectric increment of the isoionic solution, is found to be a sensitive, monotonic indicator of the number of moles (up to at least 5) of long chain fatty acid complexed. The sensitivity is about three times as great as it is in bovine albumin. New methods of analysis of the frequency dispersion of the dielectric constant were developed to ascertain if molecular shape changes also accompany the complexing with fatty acid. Direct two-component rotary diffusion constant analysis is found to be too strongly affected by cross modulation between small systematic errors and physically significant data components to be a reliable measure of structural modification. Multicomponent relaxation profiles are more useful as recognition patterns for structural comparisons, but the equations involved are ill-conditioned and solutions based on standard least-squares regression contain mathematical artifacts which mask the physically significant spectrum. By constraining the solution to non-negative coefficients, the magnitude of the artifacts is reduced to well below the magnitudes of the spectral components. Profiles calculated in this way show no evidence of significant dipole direction or molecular shape change as the albumin is complexed with 1 mol of fatty acid. In these experiments albumin was defatted by incubation with adipose tissue at physiological pH, which avoids passing the protein through the pH of the N-F transition usually required in defatting. Addition of fatty acid from soluion in small amounts of ethanol appears to form a complex indistinguishable from the "native" complex.