Intrinsic Fluorescence Spectroscopy Research Articles

Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme.

Amino-terminal and carboxy-terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452). The wild-type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L-Phe) binding (Hill coefficient h = 2.2, S0.5 = 154 microM). The deletion mutants lacking the carboxy-terminal 24 amino acids hPAH (Ser2-Gln428) and hPAH(Gly103-Gln428) formed catalytically active dimers, and incubation with L-Phe did not promote the formation of tetramers, a characteristic property of dimeric wt-hPAH. The carboxyterminus thus seems to contain a motif required for dimer-dimer interaction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Ser2-Gln428) and hPAH(Gly103-Gln428) were all activated by prior incubation with L-Phe, but did not reveal any positive cooperativity of substrate binding (h = 1.0). The activation by L-Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino-terminal sequence. i.e. wt-hPAH and the Ser2-Gln428 mutant. The amino-terminal deletion mutants hPAH(Asp112-Lys452) and hPAH(Gly103-Gln428) revealed high specific activity, increased apparent affinity for L-Phe (S0.5 = 60 microM) and a tryptophan fluorescence emission spectrum similar to that of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt-hPAH amino-terminal sequence. Our results are compatible with a model in which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino-terminal domain to the high-affinity binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.

Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.

Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alpha 1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.

Hb Montefiore (α126(H9)Asp → Tyr) HIGH OXYGEN AFFINITY AND LOSS OF COOPERATIVITY SECONDARY TO C-TERMINAL DISRUPTION

Hb Montefiore was found, in the heterozygous state, in a Puerto Rican female who had a slightly elevated total Hb level. Structural analysis revealed that Asp-alpha126 was replaced by Tyr. Hb Montefiore migrates close to HbF (at pH 8.6) and accounts for 20.3% of the hemolysate. Oxygen binding of red blood cells revealed a 40% decrease in the P50 (pH 7.4) and a low n value of 1.6 (normal: 2.6). Depletion of red blood cell 2,3-DPG did not change the results. Stripped Hb Montefiore at pH 7.2 showed an 8-fold reduction in P50 (0.6 versus 4.6 mm Hg) and very low cooperativity (n = 1.2 versus 2.9 for the control). Heterotopic effectors, as 2,3-diphosphoglycerate and inositol hexaphosphate had a normal effect and in addition, they increased cooperativity. The chloride ion effect and the Bohr effect were moderately reduced. A bezafibrate derivative (L345), known to bind alpha126, increases the P50 of HbA by 9-fold, but only by 1. 5-fold that of Hb Montefiore. Combining these functional studies with intrinsic fluorescence and Resonance Raman spectroscopy, we interpret the very low n value and the high oxygen affinity for Hb Montefiore as a result of both a destabilized T state that switches to R upon ligand binding and a deoxy T state that binds ligands with higher affinity than that of deoxy HbA. Hb Montefiore still binds ligands cooperatively, but the difference in ligand binding properties of the two quaternary states has been drastically reduced.

Expression of Biologically Active Human SPARC inEscherichia coli

Human SPARC has been cloned by the polymerase chain reaction from an endothelial cell cDNA library and expressed inEscherichia colias a biologically active protein. Transcriptional expression of the insert cDNA was dependent on the activation of the T7 RNA polymerase promoter by isopropylgalactopyranoside. Two forms of recombinant SPARC (rSPARC) protein were recovered from BL21(DE3)E. coliafter transformation with the plasmid pSPARCwt: a soluble, monomeric form of rSPARC and an insoluble, aggregated form sequestered in inclusion bodies. The isolation of the soluble form of rSPARC was accomplished by anion-exchange, nickel-chelate affinity, and gel filtration chromatographies. The isolated protein was an intact, full-length polypeptide of 293 amino acids by the following criteria: N-terminal amino acid sequence, reaction with anti-SPARC immunoglobulins specific for N-terminal and C-terminal sequences, and interaction of the C-terminal histidine tag of rSPARC with a nickel-chelate affinity resin. Circular dichroism and intrinsic fluorescence spectroscopy indicated that the conformation of rSPARC was dependent on interaction with Ca2+ions. The recombinant protein inhibited cell spreading and bound specifically to bovine aortic endothelial cells. Levels of bacterial endotoxin (<18 pg/μg rSPARC) present in rSPARC preparations were below the threshold that affects the behavior of these endothelial cells. These conformational and biological properties of rSPARC are consistent with previously described characteristics of the native protein. The purification of biologically active rSPARC, as well as mutated forms of the protein, will provide sufficient quantities of protein for the determination of structure/function relationships.

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

α-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of αA and αB subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to α-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37°C: binding sites per 400 kDa aggregate = 10 and K a = 8.1 · 10 3 M −1; and an essentially identical K a of 7.84 · 103 M −1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, all, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22°C and was absent at 4°C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05–0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum at 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P i, AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/α-crystallin binding.

Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher α-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.

Betaine counteracts urea‐induced conformational changes and uncoupling of the human erythrocyte Ca2+ pump

We have investigated the effects of the protein structure-perturbing and function-perturbing osmolyte urea, and one of its physiological counteracting solutes, the methylamine compound (carboxymethyl)trimethylammonium hydroxide (betaine), on the structure and function of the human erythrocyte plasma-membrane Ca(2+)-ATPase. Betaine per se promoted a conformational change in the purified ATPase as revealed by steady-state and time-resolved intrinsic fluorescence spectroscopy. The conformational change promoted by betaine was shown to be related to changes in the degree of compaction of the protein structure, as detected by fluorescence-quenching measurements using acrylamide and iodide, non-charged and charged quenchers, respectively. In contrast, urea promoted a biphasic increase in exposure of tryptophan residues of the purified ATPase to the aqueous medium. With the use of membrane-bound ATPase, increasing concentrations of urea up to 1.5 M promoted a twofold increase in the Ca(2+)-ATPase activity, and the simultaneous inhibition of Ca2+ accumulation indicated that ATP hydrolysis became uncoupled from Ca2+ transport. Higher urea concentrations promoted a pronounced inhibition of ATP hydrolysis. In the absence of urea, betaine decreased ATP hydrolysis without affecting Ca2+ transport, whereas it counteracted the strong inhibition of Ca(2+)-ATPase activity by urea concentrations as high as 7 M. Betaine also protected Ca2+ accumulation against inhibition with concentrations of urea up to 1.5 M, indicating that the methylamine is able to counteract the uncoupling of the ATPase observed at lower urea concentrations. These results suggest that betaine modifies the effects of urea on the erythrocyte Ca(2+)-ATPase, through specific solute-induced conformational changes that protect the energy-transduction capacity of the enzyme.

Detecting retinoic acid-induced biochemical alterations in squamous cell carcinoma using intrinsic fluorescence spectroscopy.

Intrinsic fluorescence spectroscopy offers a new method for diagnosing head and neck cancers. By establishing a unique spectral fingerprint for benign tissue, one can readily identify subtle changes in tissue based on altered spectral patterns. The authors applied this technology to a multicellular tumor spheroid (MTS) model and obtained baseline spectral data. A cohort of MTS was treated with the chemopreventive agent retinoic acid (RA) to determine its effect on tumor cells. Excitation and emission spectroscopy were performed on the samples. Spectroscopic scans demonstrated consistently that RA-treated MTS exhibit a decrease in the peak associated with reduced nicotinamide-adenine dinucleotide (NADH) and an increase in the peaks associated with flavins, tryptophan, and cytokeratins when compared to controls. These findings are suggestive of alterations in cellular electron transport, an increase in proteins incorporating tryptophan, and a decrease in adenosine triphosphate (ATP) in the RA-treated cells. A discussion of the potential clinical applications of intrinsic fluorescence spectroscopy is included.

Kinetic Study of the Thermal Denaturation of G Actin Using Differential Scanning Calorimetry and Intrinsic Fluorescence Spectroscopy

Previous studies of the thermal denaturation of G actin have yielded conflicting results which have led to contradictory interpretations of the denaturation process. In their interpretations, these authors postulated that the thermal denaturation of G actin can be described by thermodynamical equilibrium laws despite the fact that this transition is irreversible. Using differential scanning calorimetry and fluorescence spectroscopy, we now show that thermal denaturation of G actin does not obey to equilibrium thermodynamics, but rather, is a two-step irreversible phenomenon under kinetic control. It can therefore be analyzed using the recently developed Sanchez-Ruiz equations.

Haemocompatibility optimisation of implants by hybrid structuring

State of the art in biomaterial research and implant design is a compromise between functionality and biocompatibility. Consequently, results often have disadvantages with respect to both aspects. With regard to biocompatibility, the activation of the clotting system by alloplastic materials is of great significance, because it necessitates anticoagulant therapy. Further improvements in implant technology require an understanding of the interactions between blood and implants. Therefore a microscopic model of thrombogenesis at alloplastic surfaces is briefly presented, relating thrombogenicity of a material to the electronic structure of its surface. The electronic requirements for high haemocompatibility, which result from this model (especially a low band-gap density of states and a high surface conductivity) are fulfilled by an amorphous alloy of silicon and carbon (a-SiC:H). The advantage of amorphous materials is that they do not obey stoichiometric rules. Thus they allow a continuous adjustment of the electronic parameters without fundamental changes in their mechanical and chemical properties. The theoretical results were checked in vitro by total internal reflection intrinsic fluorescence (TIRIF) spectroscopy as well as thrombelastography experiments (TEG). In comparison with conventional materials such as titanium or LTI carbon, the TEG-clotting time of a-SiC:H-coatings was prolonged by in excess of 200 per cent. As a consequence, a-SiC:H is well suited as a haemocompatible coating material for hybrid structuring of cardiovascular implants.

Studies on Fibronectin and Its Domains: II. Secondary Structure and Spatial Configuration of Fibronectin and of Its Domains

Fibronectin (FN) from human plasma, as well as recombinant proteins derived from two of its major domains, the N-terminal fibrin-binding domain (FBD) and the cell-binding domain (CBD), were studied by circular dichroism (CD), Fourier transform infrared (FTIR), and fluorescence spectroscopies. Secondary structure estimations derived from both FTIR spectra and far uv CD indicate significant differences between FBD and CBD proteins. Thus, the CBD 33-kDa protein contains 80% β-sheet and 20% β-turns, whereas the three refolded FBD recombinant proteins studied, the 12-, 18.5-, and r31-kDa proteins, as well as the plasma-derived p31-kDa protein, all contain some percentage of aperiodic structure, ranging from 14 to 23% for both 31-kDa proteins, and between 33 and 46% for the smaller 12- and 18.5-kDa proteins. The amount of aperiodic structure increases drastically (from 23 to 67%) in a misfolded recombinant 31 kDa, the scrambled 31 kDa. Both native FN and a chimeric 45-kDa protein, consisting of both the FBD 12-kDa and the CBD 33-kDa proteins, were found to resemble the CBD 33-kDa protein itself, i.e., consisting only of β-sheet and β-turns and being free of aperiodic structure. These differences between two types of conformation are corroborated by the differences in the environment and spatial configuration of the aromatic amino acids of these proteins, as evidenced from near uv CD and intrinsic fluorescence spectroscopies. Thus, in the near uv CD the typical CBD proteins, i.e., the 33-and 45-kDa proteins display a maximum at 294 nm, whereas the FBD proteins, except for the scrambled 31-kDa protein, all have a characteristic strong negative peak at 300 nm. Similarly, the λmax of the emission spectra of the whole FN and the 33- and 45-kDa proteins are characteristically blue-shifted (317-326 nm) and with high normalized intensity, when compared with the relatively red-shifted in λmax (338-340 nm) and low intensities of the folded FBD proteins. The scrambled 31-kDa protein again takes exception, having a relatively high normalized intensity due to misfolding. The two maior conclusions from this work, attained by a combination of four spectroscopic methods, are: (a) functionally different FN domains, i.e., the FBD and CBD adopt totally different conformations, with the CBD having an almost exclusive beta structure; (b) the functional specificity of the various domains of FN may depend on subtle differences in the flexibility in their protein backbones, with the FBD being more flexible than the CBD.

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.

Conformational effects in the reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH).

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Physical mechanisms of solid-protein interactions in the interface between amorphous silicon carbide and fibrinogen

State of the art in biomaterial research and implant design is a compromise between functionality and biocompatibility. Consequently the results often have disadvantages with respect to both aspects. In regard to biocompatibility the activation of the clotting system by alloplastic materials is of great significance, because it necessitates anticoagulant therapy. Further improvements of implant technology require an understanding of the interactions between blood and implants. Therefore a microscopic model of thrombogenesis at alloplastic surfaces will shortly be presented, which relates thrombogenicity of a material to the electronic structure of its surface. The requirements for high hemocompatibility, which result from this model--especially in regard to the density of states and the conductivity at the surface--are fulfilled by an amorphous alloy of silicon and carbon (a-SiC:H). The advantage of amorphous materials is that they do not obey stoichiometric rules. Thus they allow a continuous adjustment of the electronic parameters without fundamental changes of their mechanical and chemical properties. The theoretical results where checked by total internal reflection intrinsic fluorescence spectroscopy (TIRIF) as well as thrombelastography experiments (TEG). In comparison to conventional materials like titanium or LTI carbon the TEG-clotting time of a-SiC:H-coatings is prolonged in excess of 200%. As a consequence a-SiC:H is well suited as a hemocompatible coating material for hybrid structuring of cardiovascular implants.

Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate

Serpins form a family of structurally related proteins, many of which function in plasma as inhibitors of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. To further characterize the mechanism by which serpins inhibit their target enzymes, we have studied the effect of temperature on the reaction of C1 inhibitor and the serine protease plasma kallikrein. At both 38 and 4 degrees C, C1 inhibitor (Mr 105,000) is cleaved by alpha-kallikrein (Mr 85,000 and 88,000) at position P1 (Arg444) of the reactive center, a reaction that leads to the formation of a covalent bimolecular enzyme-serpin complex (Mr 195,000) and cleaved but uncomplexed serpin (Mr 95,000). Between 38 and 4 degrees C, the product distribution is temperature-dependent, with more cleaved C1 inhibitor (Mr 95,000) formed at lower temperatures and correspondingly less Mr 195,000 complex. Studies employing intrinsic tryptophan fluorescence and 1H NMR spectroscopy show that this behavior is not caused by temperature-dependent conformational changes of kallikrein or C1 inhibitor. C1 inhibitor also behaves in this manner with the light chain of kallikrein and, to a lesser extent, with plasmin and C1s. These data are best explained by a branched reaction pathway, identical with the scheme describing the mechanism of action of suicide substrates. This scheme involves the formation of an enzyme-inhibitor intermediate, which can be stabilized into a covalent complex and/or dissociate into free enzyme and cleaved inhibitor, depending on the reaction conditions.

Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; Comparison of efficiency and specificity

Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80-fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA-antibody conjugates may be potential candidates for therapeutical use.

Noncovalent complex between domain AB and domains CD*EF of parvalbumin

The interaction between domain AB and domains CD*EF of pike parvalbumin III has been studied by intrinsic fluorescence spectroscopy. In the presence of Ca2+ ions, parvalbumin fragment 38-108 containing two calcium binding sites interacts with the short peptide 1-37 with association constant 10(5.3 +/- 0.5) M-1. Removal of Ca2+ ions results in the disappearance of the interaction. The affinity of the complex of the two fragments for calcium is 50-times higher than the affinity of the isolated fragment 38-108, but slightly lower than that of the intact protein.

Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26

Lens fiber cells are coupled by communicating junctions that comprise over 50% of their appositional surfaces. The main intrinsic protein (MIP26) of lens fibers is a 28.2 kDa protein that forms large gap junction-like channels in reconstituted systems. Previously, we have shown that Ca(++)-activated calmodulin (CaM) regulates the permeability of reconstituted MIP26 channels and changes the conformation of MIP26, as measured by intrinsic fluorescence and circular dichroism spectroscopy. Examination of the MIP26 amino acid sequence has revealed a basic amphiphilic alpha-helical segment (Pep C) on the C-terminus with residue distribution similar to that found in other CaM binding proteins. To test the interaction between the amphiphilic segment and CaM, both a 20-mer peptide and trp-substituted fluorescent analog have been synthesized and purified by HPLC. Evidence from spectrofluorometric titration shows that the Pep C binds with CaM in 1:1 stoichiometry and with a kd of approximately 10 nM. Neither Ca++ nor H+ alone affects the conformation of the Pep C. However, when mixed with CaM the Pep C undergoes both a dramatic blue-shift in tryptophan fluorescence emission, indicative of strong hydrophobic interaction, and an increase in circular dichroism absorption in the alpha-helical region. Additional fluorescence blue-shift and alpha-helical content occur when Ca++ is added to the CaM:Pep C complex.

Effect of metal ions on the unfolding kinetics of α-lactalbumin on weakly hydrophobic surfaces

The effect of metal ion [Ca 2+ and Zn(NH 3) 4 2+] on the unfolding kinetics of bovine-α-lactalbumin (α-LACT) on a weakly hydrophobic chromatographic surface (ethyl polyether phase bonded on porous silica, C 2 ether) has been studied using surface intrinsic fluorescence spectroscopy and liquid chromatography. Chromatographic results on the C 2 ether phase revealed two peaks for α-LACT, the first being the folded and the second an unfolded conformation, as determined by fluorescence spectroscopy. The retention time for the second peak was found to depend on the specific metal additive in the mobile phase.Fluorescence studies showed a slow change in emission maximum from ca. 330 nm to 350 nm and a 5-fold increase in emission intensity for the adsorbed protein in the unfolded state. By following the fluorescence emission intensity at a given wavelength during the unfolding process, biphasic kinetics were observed with the kinetic constants depending on the specific metal-ion additive. In addition, solution refolding rates of the desorbed, unfolded species were measured and found to be consistent with literature refolding rate constants.

Purification and characterization of recombinant Rev protein of human immunodeficiency virus type 1.

Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.