Intrinsic Fluorescence Measurements Research Articles

Spectrophotometric and Colorimetric Determination of Protein Concentration

AbstractSpectrophotometric and Colorimetric Determination of Protein Concentration (Michael H. Simonian, Beckman‐Coulter, Inc., Fullerton, California, and John A. Smith, University of Alabama at Birmingham, Birmingham, Alabama). Measuring absorbance at 280 nm (A280) is one of the oldest methods for determining protein concentration. This method is still widely used because it is simple and does not require incubating the sample with exogenous chromophores. Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A205) is more universally applicable. Furthermore, the absorptivity for a given protein at 205 nm is several‐fold greater than that at 280 nm. Thus, lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm. The aromatic amino acids also exhibit fluorescence emissions when excited by light in the UV range. Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information. However, with a protein standard whose aromatic amino acid content is similar to that of the sample, intrinsic fluorescence can be used for quantitation. The most frequently employed colorimetric methods for determining protein concentration are those of Bradford and Lowry.

Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure.

Parkinson's disease involves the aggregation of alpha-synuclein to form fibrils, which are the major constituent of intracellular protein inclusions (Lewy bodies and Lewy neurites) in dopaminergic neurons of the substantia nigra. Occupational exposure to specific metals, especially manganese, copper, lead, iron, mercury, zinc, aluminum, appears to be a risk factor for Parkinson's disease based on epidemiological studies. Elevated levels of several of these metals have also been reported in the substantia nigra of Parkinson's disease subjects. We examined the effect of various metals on the kinetics of fibrillation of recombinant alpha-synuclein and in inducing conformational changes, as monitored by biophysical techniques. Several di- and trivalent metal ions caused significant accelerations in the rate of alpha-synuclein fibril formation. Aluminum was the most effective, along with copper(II), iron(III), cobalt(III), and manganese(II). The effectiveness correlated with increasing ion charge density. A correlation was noted between efficiency in stimulating fibrillation and inducing a conformational change, ascribed to formation of a partially folded intermediate. The potential for ligand bridging by polyvalent metal ions is proposed to be an important factor in the metal-induced conformational changes of alpha-synuclein. The results indicate that low concentrations of some metals can directly induce alpha-synuclein fibril formation.

Molecular characterisation of size exclusion chromatography refolded urokinase-plasminogen activator

We have used five physical and structural techniques to characterise the chromatographic refolding of the serine protease fragment of recombinant human urokinase plasminogen activator (u-PA) from inclusion bodies. Sephacryl S-300 was used to refold GdnHCl-solubilised and DTT-denatured u-PA from a starting concentration of 8 mg/ ml . Dynamic light scattering results showed that protein aggregates were the first component to be eluted followed by unfolded u-PA and finally compact, folded u-PA. This was confirmed by analytical size exclusion chromatography, which also indicated that for each elution fraction, a population of u-PA species existed which varied in their hydrodynamic radius. At later elution volumes, u-PA was eluted as a monomeric, active molecule. The use of intrinsic fluorescence measurements across the elution fractions showed a correlation between stabilisation of the fluorescence signal and peak u-PA activity. Far UV circular dichroism identified the increasing formation of β-sheet structures in the refolding molecule. The use of tryptic digestion-RPLC showed identical digestion patterns between SEC and batch refolded u-PA. Finally, analysing the tryptic digestion profiles by mass spectrometry identified four peptide sequences on the surface of the u-PA molecule (IRSKEGR, VSHFLPWIR, GCALKDKPGVYTR and IIGGEFTTIENQPWF) that became more accessible to digestion as folding progressed.

Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus.

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.

Metal ions modulate the plastic nature of Mycobacterium tuberculosis chaperonin-10.

Chaperonin-10s possess a highly flexible segment of approximately 10 residues that covers their dome-like structure and closes the central cavity of the chaperonin assembly. The dome loop is believed to contribute to the plasticity of their oligomeric structure. We have exploited the presence of a single tryptophan residue occurring in the dome loop of Mycobacterium tuberculosis chaperonin-10 (cpn-10), and through intrinsic fluorescence measurements show that in the absence of metal ions, the tryptophan is almost fully solvent exposed at neutral pH. The dome loop, however, assumes a closed conformation in the presence of metal ions, or at low pH. These changes are fully reversed in the presence of chelating agents such as EDTA, confirming the role of cations in modulating the metastable states of cpn-10.

Kinetic properties of chitinase-1 from the fungal pathogen Coccidioides immitis.

The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the beta-conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc)6 is predominantly cleaved into (GlcNAc)2 and (GlcNAc)4, where the (GlcNAc)2 group arises from the nonreducing end of the substrate and is formed as the beta-anomer. With time, transglycosylation occurs, generating (GlcNAc)8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc)5 and (GlcNAc)4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc)4 is 50 microM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc)6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.

The Gla Domain of Human Prothrombin Has a Binding Site for Factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB.

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

Simultaneous light scattering and intrinsic fluorescence measurement for the classification of airborne particles

We describe a prototype laboratory light-scattering instrument that integrates two approaches to airborne particle characterization: spatial light-scattering analysis and intrinsic fluorescence measurement, with the aim of providing an effective means of classifying biological particles within an ambient aerosol. The system uses a single continuous-wave 266-nm ultraviolet laser to generate both the spatial elastic scatter data (from which an assessment of particle size and shape is made) and the particle intrinsic fluorescence data from particles in the approximate size range of 1-10-mum diameter carried in a sample airflow through the laser beam. Preliminary results suggest that this multiparameter measurement approach can provide an effective means of classifying different particle types and can reduce occurrences of false-positive detection of biological aerosols.

Thermal unfolding and refolding of β‐lactoglobulin

The conformational features of beta-lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85-90 degrees C, beta-lactoglobulin does not completely lose its folded structure. The unfolding and refolding of beta-lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native beta-lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1-anilino 8-naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded beta-lactoglobulin is observed compared to that of the native form. Our results indicate that beta-lactoglobulin, refolded after heating to above 70 degrees C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native beta-lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open.

Arginine‐based structures are specific inhibitors of cathepsin C

Novel synthetic peptide inhibitors of lysosomal cysteine proteinase cathepsin C have been designed through the use of soluble peptide combinatorial libraries. The uncovered structural inhibitory module consists of the N-terminal cluster of L-arginine residues. Its modification with D-amino acids or arginine derivatives did not increase the inhibition strength. Inhibitory potency of oligoarginines improves with the elongation of peptide chain reaching a maximum for octa-L-arginine. The oligoarginines specifically interact with the cathepsin C active site as shown by competitive-type inhibition kinetics (Ki approximately 10-5 M) and intrinsic fluorescence measurements. The inhibitory interaction of oligoarginines is established through the specific spatial contact of a net of guanidino groups in the arginine side-chains, as indicated by comparison with inhibitory action of low molecular mass guanidine derivatives (Ki approximately 10-3 M). Nonarginine polyionic compounds cannot mimic the inhibitory effect of oligoarginines. The arginine-based peptide inhibitors were selective towards cathepsin C among other cysteine proteinases tested.

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(±0.25) Å and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2.2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(±0.43) Å, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(±0.25) Å, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23.5 Å). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.

Anion-induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation.

The unfolding of human serum albumin (HSA), a multidomain protein, by urea was followed by far-UV circular dichroism (CD), intrinsic fluorescence, and ANS fluorescence measurements. The urea-induced transition, which otherwise was a two-step process with a stable intermediate at around 4.8 M urea concentration as monitored by far-UV CD and intrinsic fluorescence, underwent a single-step cooperative transition in the presence of 1.0 M KCl. The free energy of stabilization (DeltaDelta G(H2O)D) in the presence of 1 M KCl was found to be 1,090 and 1,200 cal/mol as determined by CD and fluorescence, respectively. The salt stabilization occurred in the first transition (0-5.0 M urea), which corresponded to the formation of intermediate (I) state from the native (N) state, whereas the second transition, corresponding to the unfolding of I state to denatured (D) state, remained unaffected. Urea denaturation of HSA as monitored by tryptophan fluorescence of the lone tryptophan residue (Trp(214)) residing in domain II of the protein, followed a single-step transition suggesting that domain(s) I and/or III is (are) involved in the intermediate formation. This was also confirmed by the acrylamide quenching of tryptophan fluorescence at 5 M urea, which exhibited little change in the value of Stern-Volmer constant. ANS fluorescence data also showed single-step transition reflecting the absence of accumulation of hydrophobic patches. The stabilizing potential of various salts studied by far-UV CD and intrinsic fluorescence was found to follow the order: NaClO(4) > NaSCN >Na(2)SO(4) >KBr >KCl >KF. A comparison of the effects of various potassium salts revealed that anions were chiefly responsible in stabilizing HSA. The above series was found similar to the electroselectivity series of anions towards the anion-exchange resins and reverse of the Hofmeister series, suggesting that preferential binding of anions to HSA rather than hydration, was primarily responsible for stabilization. Further, single-step transition observed with GdnHCl can be ascribed to its ionic character as the free energy change associated with urea denaturation in the presence of 1.0 M KCl (5,980 cal/mol) was similar to that obtained with GdnHCl (5,870 cal/mol).

Dissociation of human alphaB-crystallin aggregates by thiocyanate is structurally and functionally reversible.

Conformational modifications and changes in the aggregation state of human alphaB-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH4SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH4SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of alphaB-crystallin in 2 M NH4SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated alphaB-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.

Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four α–helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL, proteolytic cleavage occurred at Glu residues which would be located at the end of the first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the α-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd~ 1.2 - 1.6 µM). The total concentration of binding sites (Bmax) present on the fibrin surface was ~4-fold greater for the 12K than for the 17K (Bmaxvalues of 0.81 ± 0.09 nM, and 0.20 ± 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmaxvalues of 2.7 ± 0.3 nM and 0.20 ± 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmaxwas correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low µM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.Key words: fibrinolysis, lipoprotein(a), plasminogen activation.

Dynamic and Static Light Scattering and Fluorescence Studies of the Interactions between Lactate Dehydrogenase and Poly(ethyleneimine)

Interactions between poly(ethyleneimine) (PEI) and porcine muscle lactate dehydrogenase (LDH) were studied using static and dynamic light scattering, and intrinsic fluorescence spectroscopy. Time-related aggregation of the enzyme was reduced in the presence of the polymer. PEI of molecular weights 2000 and 25000 were found to complex with the enzyme without significant change in the particle hydrodynamic radius, i.e., the polymer is bound in a flat conformation at the enzyme surface. Interactions with the high molecular weight PEI (2.6 × 106), on the other hand, resulted in a particle of a size significantly larger than the polymer, indicating that several LDH molecules may bind within the large, branched polymer structure. Although it was not found possible to quantitatively determine the degree of binding, estimation of the binding constants by intrinsic fluorescence measurements revealed weak binding between the enzyme and the polymer.

A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening.

To develop and validate a rapid method for determining the dissociation constants with which pharmaceutical candidates and drugs bind to serum albumin and to alpha1-acid glycoprotein with the goal of deducing the extent of binding. The quenching of the intrinsic tryptophan fluorescence of serum albumin and alpha1-acid glycoprotein was monitored by spectrofluorimetry and the data were used to calculate the apparent dissociation constant. Sodium warfarin was used to probe the warfarin-binding site of serum albumin and diazepam was used to probe the benzodiazepine binding site. Additionally, the binding of sodium salicylate, phenylbutazone, sulfinpyrazone, iophenoxic acid, theophylline, chloramphenicol, acetaminophen, lithium chloride and ampicillin were also investigated. Chlorpromazine hydrochloride and imipramine hydrochloride were used as probes for alpha1-acid glycoprotein. The assays were also extended to the multiwell format. The quenching curves were fitted to the quadratic binding equation to determine the dissociation constants. Intrinsic fluorescence measurements are an excellent predictor of the drug binding to human serum albumin and to alpha1-acid glycoprotein. These measurements detect binding to the warfarin and benzodiazepine binding sites of human serum albumin. The dissociation constants estimated using the method compare favorably to the dissociation constants previously reported by Epps et al. using extrinsic fluorescence methodology, and the results correlate well with equilibrium dialysis using drug displacement endpoints. These measurements can be carried out with small samples and do not require separation of the bound and unbound species. Additionally, the proposed methods eliminate membrane separations, are not compound specific and do not require analytical chromatography or mass spectrometry for quantitation. Spectrofluorimetry may prove to be a useful method for rapidly determining the protein binding of combinatorial libraries.

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd approximately 1.2-1.6 microM). The total concentration of binding sites (Bmax) present on the fibrin surface was approximately 4-fold greater for the 12K than for the 17K (Bmax values of 0.81 +/- 0.09 nM, and 0.20 +/- 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmax values of 2.7 +/- 0.3 nM and 0.20 +/- 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmax was correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low microM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.

Lanthanide ions inhibit the activity of dihydrofolate reductase from chicken liver.

The influences of mono-, bi- and trivalent metal ions (as chloride salts) on the activity of dihydrofolate reductase (DHFR) from chicken liver have been studied to elucidate the mechanism of ion-activation of this enzyme. The results show that monovalent ions (Na+ and K+) activate DHFR at low concentration reaching a maximum activation of about 2.5 fold at 0.4-0.5 M and declining at higher concentrations. Ca2+ shows similar activation but at lower concentration, reaching a maximum at 0.1 M; activity declines with further increases in concentration. At very high concentration (> 0.4 M), Ca2+ is inhibitory. The trivalent lanthanide ions, however, show a dramatic inhibition of activity of DHFR even at very low concentration. The activity of DHFR declines to 50% of that of the control at 0.02 mM EuCl3. Intrinsic fluorescence measurements show that the ion-dependent activation in the presence of mono- and bivalent metal ions is due to the conformational changes in the protein. Energy transfer phenomenon suggests that the specific interaction of Eu3+ with Trp24 located in a loop at the active site of DHFR is responsible for the strong inhibition. The possible mechanism for the ion-inhibition is proposed and discussed.