Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation.

Cell loss in Parkinson's and Parkinson's-plus diseases is linked to abnormal, aggregated forms of the cytoplasmic protein, α-synuclein (α-syn). The factors causing α-syn aggregation may include oxidative stress, changes in protein turnover and dysregulation of calcium homeostasis, resulting in cytotoxic aggregated α-syn species. Recently, we showed that raised calcium can promote α-syn aggregation. We have now investigated the effects of raised calcium combined with oxidation/oxidative stress on α-syn aggregation both in vitro and in vivo. We treated monomeric α-syn with calcium, hydrogen peroxide or calcium plus hydrogen peroxide in vitro and used size exclusion chromatography, fluorescence correlation spectroscopy, atomic force microscopy and scanning electron microscopy to investigate protein aggregation. Our in vitro data is consistent with a cooperative interaction between calcium and oxidation resulting in α-syn oligomers. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-syn-GFP construct both with and without co-treatment with hydrogen peroxide and observed α-syn aggregation by fluorescence microscopy. Our in vivo cell culture data shows that either transient increase in intracellular free calcium or hydrogen peroxide treatment individually were able to induce significantly (P=0.01) increased 1-4µm cytoplasmic α-syn aggregates after 12h in cells transiently transfected with α-syn-GFP. There was a greater proportion of cells positive for aggregates when both raised calcium and oxidative stress were combined, with a significantly increased proportion (P=0.001) of cells with multiple (3 or more) discrete α-syn focal accumulations per cell in the combined treatment compared to raised calcium only. Our data indicates that calcium and oxidation/oxidative stress can cooperatively promote α-syn aggregation both in vitro and in vivo and suggests that oxidative stress may play an important role in the calcium-dependent aggregation mechanism.

Raised calcium promotes α-synuclein aggregate formation.

Parkinson's and Parkinson's-plus diseases are associated with abnormal, aggregated forms of the protein, α-synuclein. We have investigated the effects of calcium on α-synuclein aggregation in vitro and in vivo. We treated monomeric α-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Incubation of fluorescent-labelled monomeric α-synuclein (24h) at low concentration (10 µM) with calcium resulted in surface aggregates (1.5±0.7 µm(2)) detected by fluorescence microscopy saturating at a half-maximum calcium concentration of 80 µM, whilst incubations without calcium showed few protein aggregates. Scanning electron microscopy revealed that α-synuclein surface plaques (0.5-1 µm) form in the presence of calcium and comprise 10-20 nm globular particles. Incubation of α-synuclein at high concentration (75 µM; 6h) resulted in soluble oligomeric aggregates detected by fluorescence correlation spectroscopy in a calcium dependent process, saturating at a half maximum calcium concentration of 180 µM. In cell culture experiments, we used thapsigargin or calcium ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-synuclein-GFP construct and observed calcium flux and α-synuclein aggregation by fluorescence microscopy. The cell culture data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic α-synuclein aggregates 6 and 12h post-treatment (P, 0.01). Our data indicates that calcium accelerates α-synuclein aggregation on surfaces, in free solution and in cultured cells and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.

Chemical and biological characterization of thiol SAMs for neuronal cell attachment.

Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.

Peptide-functionalized microfabricated structures for improved on-chip neuronal adhesion.

Extracellular, high signal-to-noise ratio recordings from electrogenic cells require a tight coupling between the cellular membrane and the recording electrode. Self assembled monolayers (SAMs) of alkanethiols functionalized with peptides were used in combination with micro- and nano-structured features on the sensor surface. This combination of surface chemistry and topography triggers a phagocytosis-like engulfment and ensures tight coupling. In this paper we report the results concerning usage of different SAMs and the influence of the peptide concentration towards cell adhesion and outgrowth. Later on, the optimized peptide functionalized SAMs were applied on micro- and nano-structured sensor surfaces. As a result, phagocytosis-like events could be shown using focused ion beam SEM and confocal fluorescence imaging.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.

BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action.

OBJECTIVES: We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. METHODS: We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. RESULTS: Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. CONCLUSIONS: These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Stability analysis of the bacteriophage phiKMV lysin gp36C and its putative role during infection.

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage phiKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h(-1) at 95 degrees C), but not thermostable (T(m) = 50.2 degrees C, DeltaH(cal) = 6.86 x 10(4) cal mol(-1)). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55 degrees C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85 degrees C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection.

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Novel inhibitors of HIV-1 integration.

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.

Non-Gaussian statistics of the vibrational fluctuations of myoglobin.

Experiments on the dynamics of vibrational fluctuations in myoglobin revealed an interesting behavioral cross-over occurring in the range 180-200 K. In this temperature range the mean square displacement of atomic positions versus temperature sharply increases its slope, indicating the dissociation of CO from the heme group. In this paper we develop a theoretical model that provides a framework for the quantitative description of this phenomenon. The basis of our calculations is an assumption of an effective potential with multiple local minima. In particular, we consider a quartic potential in place of the simple quadratic. We then use non-Gaussian statistics to obtain a relationship between the mean square displacement and model parameters. We compare our model to published experimental data and show that it can describe the data set using physically meaningful parameters which are fitted to the experimental data. In the process we verify the Gaussian approximation's applicability only to the low-temperature régime. In the high-temperature limit, however, deviations from the Gaussian approximation are due to the double-well nature of our effective potential. We find that the published datasets showing the thermal transition display the qualitative trends predicted by appropriate algebraic approximations to our predicted myoglobin behavior.

The analysis of time resolved protein fluorescence in multi-tryptophan proteins.

In the last decades, considerable progress has been made in the analysis of the fluorescence decay of proteins with more than one tryptophan. The construction of single tryptophan containing proteins has shown that the lifetimes of the wild type proteins are often the linear combinations of the family lifetimes of the contributing tryptophan residues. Additivity is not followed when energy transfer takes place among tryptophan residues or when the structure of the remaining protein is altered upon the modification. Progress has also been made in the interpretation of the value of the lifetime and the linkage with the immediate environment. Probably all the irreversible processes leading to return to the ground state have been catalogued and their rate constants are documented. Also, the process of electron transfer to the peptide carbonyl is becoming more and more documented and is linked to the rotameric state of tryptophan. Reversible excited state processes are also being considered, including reversible interconversions between rotamers. Interesting information about tryptophan and its environment comes also from anisotropy measurements for proteins in the native, the denatured and the molten globule states. Alterations of protein fluorescence due to the effects of ligand binding or side chain modifications can be analyzed via the ratio of the quantum yields of the modified protein and the reference state. Using the ratio of quantum yields and the (amplitude weighted) average lifetime, three factors can be identified: (1) a change in the apparent radiative rate constant reflecting either static quenching or an intrinsic change in the radiative properties; (2) a change in dynamic quenching; and (3) a change in the balance of the populations of the microstates or local static quenching.

Interactions between oligonucleotides and cationic polymers investigated by fluorescence correlation spectroscopy.

PURPOSE: To evaluate whether fluorescence correlation spectroscopy (FCS) can be used to characterize the complexation between oligonucleotides and cationic polymers. METHODS: The features of the complexes between rhodamine labeled oligonucleotides (Rh-ONs) and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), poly(ethylene glycol)-poly(ethyleneimine) (pEG-pEI), and diaminobutane-dendrimer-(NH2)64 (DAB64) were characterized by light scattering, electrophoretic mobility, electrophoresis, and FCS. RESULTS: At low polymer/Rh-ON ratios, a decrease of the fluorescence of the Rh-ONs was observed on binding of the Rh-ONs to all cationic polymers. This was explained by the creation of "multimolecular complexes" in which the Rh-labels quench each other. The multimolecular complexes, which are highly fluorescent as they carry a number of Rh-ONs, resulted in high fluorescence peaks in the fluorescence fluctuation profile as measured by FCS. For pDMAEMA and DAB64, at higher polymer/Rh-ON ratios the fluorescence of the polyplexes increased, caused by the formation of "monomolecular complexes," which consist of only one Rh-ON per polymer. In the case of pEG-pEI, the fluorescence stayed constant when the polymer/Rh-ON ratio increased, so multimolecular polyplexes remained. FCS confirmed these results as the high fluorescence peaks disappeared in case of pDMAEMA/Rh-ON and DAB64/Rh-ON dispersions, but remained present for pEG-pEI/Rh-ON dispersions. CONCLUSIONS: FCS seems applicable for study of the interactions between ONs and different types of cationic polymers.

Membrane topology of the Streptomyces lividans type I signal peptidases.

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers.

The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (epsilon), as revealed from PCH analysis, was greater than epsilon for the free Rh-ONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

Extending the capabilities of targeted molecular dynamics: simulation of a large conformational transition in plasminogen activator inhibitor 1.

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasminogen activators such as tissue-type plasminogen activator or urokinase-type plasminogen activator. For this molecule, different conformations are known. The inhibiting form that interacts with the proteinases is called the active form. The noninhibitory, noncleavable form is called the latent form. X-ray and modeling studies have revealed a large change in position of the reactive center loop (RCL), responsible for the interaction with the proteinases, that is inserted into a beta-sheet (s4A) in the latent form. The mechanism underlying this spontaneous conformational change (half-life = 2 h at 37 degrees C) is not known in detail. This investigation attempts to predict a transition path from the active to the latent structure at the atomic level, by using simulation techniques. Together with targeted molecular dynamics (TMD), a plausible assumption on a rigid body movement of the RCL was applied to define an initial guess for an intermediate. Different pathways were simulated, from the active to the intermediate, from the intermediate to the latent structure and vice versa under different conditions. Equilibrium simulations at different steps of the path also were performed. The results show that a continuous pathway from the active to the latent structure can be modeled. This study also shows that this approach may be applied in general to model large conformational changes in any kind of protein for which the initial and final three-dimensional structure is known.

Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.

Microenvironment of cysteine 242 in type-1 ribosome-inactivating protein from iris.

IRIP is a type-1 ribosome-inactivating protein isolated from the bulbs of Iris hollandica. It is one of the few type-1 RIPs that contain Cys residue(s) in their primary sequence. IRIP contains a single Cys residue at position 242. Although IRIP is thought to be a monomeric protein, SDS-PAGE indicates that part of the IRIP molecules can exist as disulphide bridge-linked dimers. Probing of the reactivity of the unique Cys residue by 5, 5'-dithiobis(2-nitrobenzoic acid) indicates that Cys(242) in IRIP is free but is only partially accessible to modifiers. Molecular modelling of IRIP is in agreement with this conclusion. Binding of the ligands adenine and poly(A) results in little or no effect on the conformation of Cys(242) in IRIP. Chemical modification of IRIP by a specific thiol modifier does not abolish the RNA N-glycosidase activity of IRIP, suggesting that Cys(242) is not critical for the enzymatic activity of IRIP. These results suggest that IRIP has the potential to be developed as a novel immunotoxin.

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.