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Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin‐based anticoagulants such as unfractionated heparin, low‐molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA‐approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin‐binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies. A new concept in the design of universal heparin antidote based on switchable protonation of cationic ligands assembled on a polymer scaffold to allow for efficient heparin neutralization in surgery and to prevent bleeding is presented. The lead compound demonstrates superior neutralization of all clinically used heparins with high biocompatibility.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG)n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG)n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG)n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG)n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

Anticoagulants such as unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and direct oral anticoagulants (DOACs) targeting thrombin (IIa) or factor Xa (FXa) are widely used in prevention and treatment of thromboembolic disorders. However, anticoagulant-associated bleeding is a concern that demands monitoring and neutralization. Protamine, the UFH antidote, has limitations, while there is no antidote available for certain direct FXa inhibitors. Improved antidotes in development include UHRA (Universal Heparin Reversal Agent) for all heparin anticoagulants; andexanet alfa (andexanet), a recombinant antidote for both direct FXa inhibitors and LMWHs; and ciraparantag (PER977), a small-molecule antidote for UFH, LMWHs, and certain DOACs. The binding affinities of these antidotes for their presumed anticoagulant targets have not been compared. Here, isothermal titration calorimetry (ITC) was used to determine the affinity of each antidote for its putative targets. Clotting and chromogenic FXa assays were used to characterize neutralization activity, and electron microscopy was used to visualize the effect of each antidote on clot morphology in the absence or presence of anticoagulant. ITC confirmed binding of UHRA to all heparins, and binding of andexanet to edoxaban and rivaroxaban, and to the antithrombin–enoxaparin complex. PER977 was found to bind heparins weakly, but not the direct FXa inhibitors studied. For UHRA and andexanet, an affinity at or below the micromolar level was found to correlate with neutralization activity, while no reversal activity was observed for the PER977/anticoagulant systems. Standard metrics of clot structure were found to correlate weakly with PER977’s activity. This is the first study comparing 3 antidotes in development, with each exerting activity through a distinct mechanism.

Anticoagulants are widely used for the prophylaxis and treatment of cardiovascular disorders and to prevent blood clotting during surgeries. However, the major limitation associated with anticoagulant therapy is bleeding; all the current anticoagulants do have a bleeding risk. The propensity to bleed is much higher among the elderly population and patients with renal insufficiency. Therefore, there is an utmost and urgent clinical need for a highly efficient, nontoxic antidote with excellent anticoagulant reversal activity. This will significantly improve the safety of anticoagulation therapy. This review summarizes the current options and approaches to reverse anticoagulation activity of clinically used anticoagulants. We start with an introduction to thrombosis and then summarize the details of current clinically available anticoagulants and their mechanisms of action and limitations. This is followed by current practices in anticoagulant neutralization including the details of the only clinically approved unfractionated heparin antidote, protamine; recent advances in the development of antidotes against heparin-based drugs; and direct oral anticoagulants (DOACs).

Anticoagulant therapy–associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

Anticoagulant therapy–associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAFV600Ebut also all known somatic point mutations within the BRAFV600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAFand a novel wild-type–negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

This article evaluates a threefold approach for assessing cultural and linguistic diversity (CLD) content in the core curriculum of a speech-language pathology master’s program. A standard syllabus review was supplemented with individual faculty interviews and a student-generated review of course content. The data from the three approaches were entered into a CLD Knowledge and Skills Matrix. Results revealed that the syllabus review did not sufficiently document CLD course content; the faculty interview and course content review provided additional perspectives necessary to interpret CLD content coverage and instructional methods. Implications for assessing CLD curricular infusion to support development of cultural competency are discussed.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP’s prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750UHRA-8 and 32 in mPEG750UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

The article discusses the conflict between sexual orientation and religious beliefs in public educational institutions. The author explains recognition of rights usually depends on the geographical location as there are conservative and liberal states. The document "Harassment, Bullying and Free Expression: Guidelines for Free and Safe Schools," helps administrators find equilibrium. He concludes teachers should help students adhere to civil discourse.

Noninvasive delivery of macromolecules across intact skin is challenging but would allow for needle-free administration of many pharmaceuticals. Biphasic vesicles, a novel lipid-based topical delivery system, have been shown to deliver macromolecules into the skin. Investigation of the delivery mechanism of interferon alpha (IFN α), as a model protein, by biphasic vesicles could improve understanding of molecular transport through the stratum corneum and allow for the design of more effective delivery systems. The interaction of biphasic vesicles with human skin and isolated stratum corneum membrane was investigated by confocal microscopy, differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS and WAXS). Confocal microscopy revealed that biphasic vesicles delivered IFN α intercellularly, to a depth of 70 μm, well below the stratum corneum and into the viable epidermis. DSC and SAXS/WAXS data suggest that the interaction of biphasic vesicles with SC lipids resulted in the formation of a three-dimensional cubic Pn3mpolymorphic phase by the molecular rearrangement of intercellular lipids. This cubic phase could be an intercellular permeation nanopathway that may explain the increased delivery of IFN α by biphasic vesicles. Liposomes and submicrometer emulsion (the individual building blocks of biphasic vesicles) separately and methylcellulose gel, an alternative topical vehicle, did not induce a cubic phase and delivered low amounts of IFN α below the stratum corneum. Molecular modeling of the cubic Pn3mphase and lamellar-to-cubic phase transitions provides a plausible mechanism for transport of IFN α. It is hypothesized that induction of a Pn3mcubic phase in stratum corneum lipids could make dermal and transdermal delivery of other macromolecules also possible.

The article focuses on the need to teach students about the importance of community and citizenship. It explains that most schools would claim to encourage civic service and discusses the traits that exemplify civic character, such as honesty, respect for others, and political participation. Fairview Elementary School in Modesto, California, is examined for its community and parent activities, and other schools with active community programs are also noted.

The author argues that school districts should develop comprehensive policies related to religious expression. According to the author, school districts should be proactive in designing policies regarding religion and schools. The article discusses the various issues related to religion in schools including: Bible studies electives; religious discussion in history classes; and prayer in public schools. INSET: Taking Religion Seriously in the Curriculum.

Reservoir characterization, modeling, and simulation were undertaken to improve production from Womack Hill field (eastern Gulf Coast, United States). This field produces oil from Upper Jurassic Smackover carbonate shoal reservoirs. These reservoirs occur in vertically stacked, heterogeneous depositional and porosity cycles. The cycles consist of lime mudstone and wackestone at the base and ooid grainstone at the top. Porosity has been enhanced through dissolution and dolomitization. Porosity is chiefly interparticle, solution-enlarged interparticle, grain moldic, intercrystalline dolomite, and vuggy pores. Dolostone pore systems and flow units have the highest reservoir potential. Petroleum-trapping mechanisms include a fault trap (footwall uplift with closure to the south against a major west-southeast-trending normal fault) in the western area, a footwall uplift trap associated with a possible southwest-northeast-trending normal fault in the south-central area, and a salt-cored anticline with four-way dip closure in the eastern area. Potential barriers to flow are present as a result of petrophysical differences among and within the cycles, as well as the presence of normal faulting. Reservoir performance analysis and simulation indicate that the unitized western area has less than 1 MMSTB of oil remaining to be recovered, and that the eastern area has 2-3 MMSTB of oil to be recovered. A field-scale reservoir management strategy that includes the drilling of infill wells in the eastern area of the field and perforating existing wells in stratigraphically higher porosity zones in the unitized western area is recommended for sustaining production from the Womack Hill field. [ABSTRACT FROM AUTHOR]

Discusses the relevance of school communities in shaping the culture of U.S. citizens. Relationship between democracy and public education; Renewal of the civic mission of public schools; Information on a national reform initiative designed to help schools teach and practice democratic principles throughout the community. INSET: Getting Involved.

Competition isotherms are used to identify the set of cellulose substructures to which cellulose binding modules (CBMs) from families 2a, 3, 4, 9, and 17 bind. The experiments are based on coupling a unique fluorescent tag to each CBM in a manner that does not alter the natural binding properties of the CBM and therefore allows the surface and solution concentrations of each CBM to be monitored as a function of time and composition. Adsorption and surface exchange of like or competing CBMs are monitored using a range of cellulose preparations varying in both crystallinity and provenance. CBMs from families 2a, 3, 4, 9, and 17 are shown to recognize different physical forms of prepared cellulose. The demonstration of the very fine binding specificity of cellulose-specific CBMs implies that the polysaccharide targets of CBMs extend down to the resolution of cellulose microstructures.

In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni²⁺-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains ~80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >10⁴. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 724–732, 2002.

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 806–814, 2002.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir–CesT and enzymatic activity profiles of SigD–SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

Excess chemical potential, μ1PE, partial molar enthalpy, H1PEand volume of 1-propanol, V1PE, were determined as a function of mole fraction of 1-propanol, x1P, in mixed solvents of aqueous 2-propanol with various initial mole fraction of 2-propanol, x2P0. The 1-propanol–1-propanol interaction functions, H1P–1PE≡ N(∂H1PE/∂n1P), and V1P–1PE≡ N(∂V1PE/∂n1P), were evaluated by graphical differentiation. The x1P-dependence of all these quantities indicates that 1-propanol and 2-propanol modify the molecular organization of H2O in the same and additive manner in the water-rich region. The additive effect of 1-propanol and that of 2-propanol are in the ratio of (0.07/0.08).

We measured vapour pressures of aqueous acetonitrile (abbreviated as ACN) at 6, 20, and 37°C, from which excess chemical potentials of ACN (µ EACN) were calculated. We also determined excess partial molar enthalpies of ACN (HEACN) at 6, 20, 30, 37, and 45°C. From these data, excess partial molar entropies of ACN (SEACN) were calculated at 6, 20, and 37°C. Using density data by Benson's group, excess partial molar volumes of ACN (VEACN) were evaluated. The response function data by the same group were also used to evaluate amplitude and wavelength of mean-square fluctuations in terms of volume, entropy, and cross between volume and entropy. All the above quantities and their dependence on the mol fraction of solute, i.e., the effect of additional solute on the above quantities were used to study the effect of acetonitrile on the molecular organization of H2O. It was found that acetonitrile works as a stronger structure-making solute than methanol. Rather its effect on H2O is about the same as that of propan-1-ol.Key words: aqueous acetonitrile, interaction functions, fluctuations.

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

We determined excess chemical potential, partial molar enthalpy, entropy and volume, heat capacity, isothermal compressibility and thermal expansivity for aqueous butan-1-ol in the water-rich region up to the phase separation boundary at 25°C. The latter three response functions were used to calculate the mean-square fluctuation densities, which signifies the amplitude, or the intensity, of fluctuations in volume, entropy or cross (entropy–volume) fluctuations. Furthermore, we calculated the (mean-square) normalized fluctuations that are indicative of the wavelength, or the extensity, as well as the amplitude of respective fluctuations. The behaviour of these thermodynamic quantities were compared with those obtained earlier in this laboratory for aqueous methanol, ethanol, propan-1-ol, tert-butanol (tert-butyl alcohol), and 2-butoxyethanol. We conclude that in the water-rich region of aqueous butan-1-ol, mixing scheme I is operative as in other alcohols, whereby butan-1-ol molecules enhance the hydrogen bond network of H₂O in their immediate vicinities with concomitant reduction of hydrogen bond probability in the bulk H₂O away from solutes. However, before reaching the phase separation boundary there was no signature indicative of the transition of mixing scheme observed in other aqueous alcohols. Thus, in aqueous butan-1-ol phase separation occurs directly from mixing scheme I, without going through mixing scheme II, which we argued earlier to be a preparation stage for phase separation for the other alcohols.

Focuses on the Establishment clause of the United States Constitution's First Amendment, which served as an experiment in freedom of religion.

The strain ofSerratia marcescensQM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-β-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of chitin and pretreatment of chitin source were investigated. Also, the effect of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and, ball-milled with a nominal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30°C.

Structural perturbations and thermodynamic-stability changes in two similar-sized globular proteins, hen egg-white lysozyme and bovine milk α-lactalbumin, upon physical adsorption to either microspheres of a negatively charged polystyrene (PS^-) latex or a dispersion of variably charged hematite (α-Fe₂O₃) are determined from differential scanning microcalorimetry (micro-DSC), isothermal titration microcalorimetry, and more conventional electrophoretic-mobility and adsorption-isotherm data. Heat of adsorption data at pH 7 for α-lactalbumin on PS^- indicate that adsorption is driven by entropic forces. Differential heat capacity data indicate that sorbent and protein-surface dehydration provide a substantial entropic driving force for adsorption. Both proteins are largely denatured on the hydrophobic PS^- surface. In contrast, lysozyme loses only a fraction of its ordered secondary structure when adsorbed to α-Fe₂O₃ while α-lactalbumin denatures almost completely upon adsorption to this hydrophilic surface. This latter difference in adsorbed-state structures is consistent with the significantly larger native-state structural stability of lysozyme as measured by micro-DSC. Kinetic analysis of adsorbed-protein micro-DSC data suggests that adsorbed lysozyme maintains a relatively high internal cohesion. Copyright 1995, 1999 Academic Press

The ability of ultraviolet resonance Raman spectroscopy (UVRRS) to determine structural, environmental, and analytical information concerning low-concentration aqueous biomolecules makes it a powerful bioanalytical and biophysical technique. Unfortunately, its utility has been limited by experimental requirements that preclude in situ or in vivo studies in most cases. We have developed the first high-performance fiber-optic probes suitable for long-term use in pulsed UVRRS applications in the deep- UV (DUV, 205–250 nm). The probes incorporate recently developed improved ultraviolet (IUV) fibers that do not exhibit the rapid solarization and throughput decay that previously hampered the use of optical fibers for delivering pulsed, DUV light. A novel 90° mirrored collection geometry is used to overcome the inner-filtering effects that plague flush-probe geometries. The IUV fibers are characterized with respect to their efficacy at transmitting pulsed, DUV laser light, and prototype probes are used to obtain pulsed UVRRS data of aromatic amino acids, proteins, and hormones at low concentrations with 205–240-nm pulsed excitation. Efficient probe geometries and fabrication methods are presented. The performance of the probes in examining resonance-enhanced Raman signals from absorbing chromophores is investigated, and the optimal excitation wavelength is shown to be significantly red-shifted from the maximum of the resonance Raman enhancement profile. Generally applicable procedures for determining optimal experimental conditions are also introduced.

The surface diffusion rate of bacterial cellulases from Cellulomonas fimion cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-β-1–4-glycanase (Cex), an endo-β-1–4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 × 10−11to a maximum of 1.2 × 10−10cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimicellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.