PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.     

Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1

Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure.     

Cys34-Cysteinylated Human Serum Albumin Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.     

Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.     

Implications of the near-planar solution structure of human myeloma dimeric IgA1 for mucosal immunity and IgA nephropathy

IgA is unique in being able to form a diverse range of polymeric structures. Increases in the levels of dimeric IgA1 (dIgA1) in serum have been implicated in diseases such as IgA nephropathy. We have determined the solution structure for dIgA1 by synchrotron x-ray and neutron scattering and analytical ultracentrifugation. The Guinier radius of gyration (RG) of 7.60-8.65 nm indicated that the two monomers within dIgA1 are arranged in an extended conformation. The distance distribution curve P(r) gave an overall length (L) of 22-26 nm. These results were confirmed by the sedimentation coefficient and frictional ratio of dIgA1. Constrained scattering modeling starting from the IgA1 monomer solution structure revealed a near-planar dimer structure for dIgA1. The two Fc regions form a slightly bent arrangement in which they form end-to-end contacts, and the J chain was located at this interface. This structure was refined by optimizing the position of the four Fab regions. From this, the best-fit solution structures show that the four Fab Ag-binding sites are independent of one another, and the two Fc regions are accessible to receptor binding. This arrangement allows dIgA1 to initiate specific immune responses by binding to FcalphaRI receptors, while still retaining Ag-binding ability, and to be selectively transported to mucosal surfaces by binding to the polymeric Ig receptor to form secretory IgA. A mechanism for the involvement of dIgA1 oligomers in the pathology of IgA nephropathy is discussed in the light of this near-planar structure.     

Cysteine residues required for the attachment of the light chain in human IgA2

In humans, there are two subclasses of IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1), IgA2m(2) and IgA2(n). In IgA1, Cys(133) in C(H)1 forms the disulfide bond to the L chain. Our previous studies indicated that in IgA2 lacking Cys(133), a disulfide bond forms between the alpha-chain and the L chain when Cys(220) is followed by Arg(221), but not when Cys(220) is followed by Pro(221), suggesting that the Cys in C(H)1 might be involved in disulfide bonding to the L chain. However, here we show that covalent assembly of the H and L chains in IgA2(n) requires hinge-proximal Cys(241) and Cys(242) in C(H)2 and not Cys(196) or Cys(220) in C(H)1. Using pulse-chase experiments, we have demonstrated that wild-type IgA2(n) with Arg(221) and Cys(241) and Cys(242) assembles through a disulfide-bonded HL intermediate. In contrast, the major intermediate for IgA2 m(1) with Pro(221) assembly was H(2) even though both Cys(241) and Cys(242) were present. Only a small fraction of IgA2 m(1) assembles through disulfide-bonded HL. Overall, our studies indicate that for IgA2 covalent assembly of the H and L chains requires the hinge-proximal cysteines in C(H)2 and that the structure of C(H)1 influences the efficiency with which this covalent bond forms.     

The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.     

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone and its interaction with human serum albumin

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone (α1) with high stereoselectivity and good yield was developed. The feature of its binding to human serum albumin (HSA) was also investigated under simulative physiological conditions via fluorescence and UV-vis absorption spectroscopy and molecular modeling methods. The results revealed that α1 caused the fluorescence quenching of HSA by the formation of α1-HSA complexes. Hydrophobic interactions played a major role in stabilizing the complex, which was in good agreement with the results of the molecular modeling study. In addition, the effect of common ions on the binding constants of α1-HSA complexes at room temperature was also discussed. All the experimental results and theoretical data indicated that α1 bound to HSA and was effectively transported and eliminated in the body. Such findings may provide useful guidelines for further drug design.     

The partly folded back solution structure arrangement of the 30 SCR domains in human complement receptor type 1 (CR1) permits access to its C3b and C4b ligands

Human complement receptor type 1 (CR1, CD35) is a type I membrane-bound glycoprotein that belongs to the regulators of complement activity (RCA) family. The extra-cellular component of CR1 is comprised of 30 short complement regulator (SCR) domains, whereas complement receptor type 2 (CR2) has 15 SCR domains and factor H (FH) has 20 SCR domains. The domain arrangement of a soluble form of CR1 (sCR1) was studied by X-ray scattering and analytical ultracentrifugation. The radius of gyration R_G of sCR1 of 13.4(±1.1) nm is not much greater than those for CR2 and FH, and its R_G/R₀ anisotropy ratio is 3.76, compared to ratios of 3.67 for FH and 4.1 for CR2. Unlike CR2, but similar to FH, two cross-sectional R_G ranges were identified that gave R_XS values of 4.7(±0.2) nm and 1.2(±0.7) nm, respectively, showing that the SCR domains adopt a range of conformations including folded-back ones. The distance distribution function P(r) showed that the most commonly occurring distance in sCR1 is at 11.5 nm. Its maximum length of 55 nm is less than double those for CR2 or FH, even though sCR1 has twice the number of SCR domains compared to CR2 Sedimentation equilibrium experiments gave a mean molecular weight of 235 kDa for sCR1. This is consistent with the value of 245 kDa calculated from its composition including 14 N-linked oligosaccharide sites, and confirmed that sCR1 is a monomer in solution. Sedimentation velocity experiments gave a sedimentation coefficient of 5.8 S. From this, the frictional ratio (f/f₀) of sCR1 was calculated to be 2.29, which is greater than those of 1.96 for CR2 and 1.77 for FH. The constrained scattering modelling of the sCR1 solution structure starting from homologous SCR domain structures generated 5000 trial conformationally randomised models, 43 of which gave good scattering fits to show that sCR1 has a partly folded-back structure. We conclude that the inter-SCR linkers show structural features in common with those in FH, but differ from those in CR2, and the SCR arrangement in CR1 will permit C3b or C4b to access all three ligand sites.     

A concise approach to 1,11-didechloro-6-methyl-4'-O-demethyl rebeccamycin and its binding to human serum albumin: fluorescence spectroscopy and molecular modeling method

1,11-Didechloro-6-methyl-4'-O-demethyl rebeccamycin (JDC-108), a rebeccamycin analog possessing potent anti-tumor activities, was prepared via a concise one-pot strategy in good yield. The interaction between JDC-108 and human serum albumin (HSA) was studied by spectroscopic methods including fluorescence spectroscopy, UV-vis absorption spectrum, and molecular modeling. The quenching mechanism of fluorescence of HSA by JDC-108 was discussed. The number of binding sites n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters DeltaH, DeltaG, DeltaS at different temperatures were calculated and the results indicated the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the reaction. The distance r between donor (HSA) and acceptor (JDC-108) was obtained according to F?rster theory of non-radiation energy transfer. Synchronous fluorescence and UV-vis absorption spectrum were used to investigate the molecular conformation of HSA molecules with addition of JDC-108 and the result indicated that molecular conformation of HSA molecules was changed in the presence of JDC-108 and the hydrophobic interaction played a major role in JDC-108-HSA association, which was in good agreement with the results of molecular modeling study. In addition, the effect of common ions on the binding constants of JDC-108-HSA complex was also discussed.     

Synthesis and characterization of protein and polylysine conjugates of sulfamethoxazole and sulfanilic acid for investigation of sulfonamide drug allergy

Conjugates of sulfamethoxazole (SMX) with human serum albumin (HSA), transferrin (TR), and poly(L-lysine) (PL, degrees of polymerization 16 and 430) have been prepared. As a model, succinylSMX-glycine methyl ester was synthesized by carbodiimide and active ester routes. The proteins and PL were acylated with succinylSMX succinimido ester, affording conjugates (succinylSMX)2-21-HSA, (succinylSMX)17,27-TR, (succinylSMX)11-Lys16, and (succinylSMX)71-Lys430 in which SMX was linked by a spacer chain of four carbons. This represents substitution of up to 35, 46, 65, and 17% of the amino groups of HSA, TR, PL16, and PL430, respectively. HSA was also acylated with the succinimido esters of succinylSMX-glycine and succinylSMX-epsilon-aminohexanoic acid, affording conjugates (succinylSMX-Gly)53-HSA and (succinylSMX-epsilon-NH2hex)51-HSA. In these conjugates SMX was linked by a spacer chain of 7 and 11 carbons, respectively, and almost all the amino groups of HSA were substituted. Factors apparently influencing the extent of conjugation to HSA were the stability of the active ester and the solubility of the conjugation reaction mixture. A sulfanilic acid (SA) conjugate, containing 12 mol of ligand/mol of HSA, was also prepared. The route of synthesis involved acylation of HSA with sulfanilyl fluoride. N-epsilon-Sulfanilyl-L-lysine dihydrochloride, required for quantitation of bound SA, was synthesized by a new route starting from alpha-Boc-L-lysine. Conjugates (sulfanilyl)12-HSA and (succinylSMX)13-HSA, differing in molecular weight from HSA by only 2.6 and 6.5%, were distinguishable from HSA by gel-filtration HPLC, as were the more highly substituted conjugates from their respective unsubstituted materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of CJC-1131-albumin bioconjugate as a stable and bioactive GLP-1(7-36) analog

A series of analogs of GLP-1(7-36) amide containing a Nepsilon-(2-[2-[2-(3-maleimidopropylamido)ethoxy]ethoxy]acetyl)lysine has been synthesized and the resulting derivatives were bioconjugated to Cys34 of human serum albumin (HSA). The GLP-1-HSA bioconjugates were analyzed in vitro to assess the stabilizing effect of bioconjugation in the presence of DPP-IV as well as GLP-1 receptor binding and activation. Compound 9 (CJC-1131) having the point of attachment to albumin at the C-terminal of GLP-1 and a D-alanine substitution at position 8 was identified as having the best combination of stability and bioactivity.     

Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris

Interleukin-2 (IL-2) plays important roles in variety of immune functions. Recombinant IL-2 has become an important therapeutic protein for therapy of melanoma and renal cell carcinoma. Previously, it was proved that the therapeutic efficacy of rIL-2 expressed in Saccharomyces cerevisiae was improved by prolonging its in vivo half-life through genetic fusion with albumin. In this study, a fusion protein composed of hIL-2 genetically fused to HSA was expressed as a secretory protein under AOX1 (alcohol oxidase 1) promoter in Pichia pastoris. An effective strategy was established to express rhIL-2-HSA fusion protein in 5L scale and the optimal purification procedure was investigated. The purity of rhIL-2-HSA in final product was about 95%. The purified rhIL-2-HSA fusion protein could be recognized by both anti-hIL-2 and anti-human serum albumin monoclonal antibody. Bioactivity analysis showed that the purified rhIL-2-HSA fusion protein displayed high level activity on proliferation in IL-2 dependent manner in CTLL2-cells. rhIL-2-HSA fusion protein also showed a extended half-life in plasma compared with IL-2 when tested in a BALB/c mouse model. This study provides an alternative strategy for large-scale production of bioactive rhIL-2-HSA fusion protein using P. pastoris as an expression host.     

Studies on the interaction between Ag(+) and human serum albumin

The interaction between Ag(+) and human serum albumin (HSA) has been intensively studied by means of equilibrium dialysis, ligand-to-metal charge transition (LMCT) bands, circular dichroism (CD) and Raman spectroscopy. Scatchard analysis of the results of equilibrium dialysis indicates the presence of two types of binding sites for Ag(+) on HSA, and the orders of magnitude of binding stability constants are found to be 10(5) and 10(4), respectively. During the binding process, a gradual increase in absorbance values of LMCT bands is observed with time-scanning UV absorption spectra, implying the Ag(I) centers are continually formed in HSA. The time-scanning CD spectra provide evidence that the binding of Ag(+) induces HSA to undergo a slow rearrangement of tertiary structure, and to change from the original conformation in the absence of Ag(+) (B-state) to conformation binding with Ag(+) (A-state). The rate constants and activation free energy of A-B transition are calculated. The Raman spectrum of Ag(I)-HSA system shows distinct vibration bands at 224 and 246 cm(-1) in the low-frequency region, which significantly reveal the formation of Ag-S and Ag-N bonds. In addition, the electrostatic interaction between Ag(+) and negatively charged oxygen is also detected with Raman spectroscopy.     

Bilirubin,model membranes and serum albumin interaction: The influence of fatty acids

Electronic circular dichroism (ECD), absorption and fluorescence spectroscopy were used to study the enantioselective interactions which involved bilirubin (BR), liposomes, human serum albumin of two different purities, pure (HSA) and non-purified of fatty acids (FA-HSA), and individual fatty acids.The application of the ECD technique to such a complex problem provided a new perspective on the BR binding to liposomes. Our results demonstrated that in the presence of pure HSA, BR preferred to bind to the protein over the liposomes. However, in the presence of FA-HSA, BR significantly bound to the liposomes composed either of DMPC or of sphingomyelin and bound only moderately to the primary and secondary binding sites of FA-HSA even at high BR concentrations. For the DMPC liposomes, even a change of BR conformation upon binding to the primary binding site was observed. The individual saturated fatty acids influenced the BR binding to HSA and liposomes in a similar way as fatty acids from FA-HSA. The unsaturated fatty acids interacted with BR alone and prevented it from interacting with either 99-HSA or the liposomes. In the presence of arachidonic acid, BR interacted enantioselectively with the liposomes and only moderately with 99-HSA.Hence, our results show a substantial impact of the liposomes on the BR binding to HSA. As a consequence of the existence of fatty acids in the blood plasma and in the natural structure of HSA, BR may possibly bind to the cell membranes even though it is normally bound to HSA.     

The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling

Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 % 2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RG from Guinier analyses were similar at 6.11-6.20 nm for IgA1 and 5.84-6.16 nm for PTerm455, and their cross-sectional radii of gyration RXS were also similar. The similarity of the RG and RXS values suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RG values are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7-9 nm and 6-8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.     

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.     

罗丹明类荧光分子探针与血清白蛋白之间相互作用的研究

本研究旨在对罗丹明类荧光探针ZM-6与人血清白蛋白(HSA)的相互作用进行研究。采用了荧光光谱法、三维荧光光谱法、同步荧光光谱法以及CD光谱法在模拟生理条件下对二者的相互作用以及HSA的构象进行了研究。研究结果表明,探针与ZM-6之间的猝灭机理主要是静态猝灭方式。根据热力学数据确定了二者之间的作用力,类型为范德华力和氢键。二者之间的结合距离为4.45 nm。同时得出,ZM-6对HSA的构象产生了影响。此研究对于探针分子的设计以及修饰提供有效的数据以及理论支持。     

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: A comparative study of the two and one tryptophan(s) of bovine and human albumins

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (_max) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the _max of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The _max of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecyltrimethylammonium bromide, the _max positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.     

Influence of direct electric current on hydrodynamic diameter of human serum albumin

The effect of the weak electric current (2 mA/cm2) on structural characteristics (hydrodynamic diameter and molecular weight) of the human serum albumin (HSA) was studied using photon correlation spectroscopy (PCS). The average diameter of initial HSA globule is approximately 7 nm (66.8 kDa). After electric current treatment during 2-5 min the diameter of HSA monomer increases to 7.5 nm. The duration of electric current treatment being increased to 20 min the size of HSA monomers decreases to 6.4 nm. The behaviour of HSA oligomers is close to that of monomers. Consequently, changes in the sizes of monomers and oligomers of HSA under the electric current treatment are caused by the change in the charge density stimulating change of tertiary structure of molecules and possible addition of ions from the buffer solution to them.