Publications

Nitric Oxide-releasing microparticles as a potent antimicrobial therapeutic against chronic rhinosinusitis bacterial isolates.

Abstract

Background: Bacteria, particularly in the biofilm state, may be implicated in the pathogenesis of chronic rhinosinusitis (CRS) and enhance antibiotic resistance. Nitric oxide (NO) is a gaseous immunomodulator with antimicrobial activity and a short half-life, complicating achievement of therapeutic concentrations. We hypothesized that a novel microparticle-based delivery platform, which allows for adjustable release of NO, could exhibit potent antibacterial effects. Methods: Porous organosilica microparticles (SNO-MP) containing nitrosylated thiol groups were formulated. Dissociation of the nitrosothiol groups generates NO at body temperature.

 

The susceptibility of bacterial isolates from CRS patients to SNO-MP was evaluated through a colony forming unit (CFU) assay. Serial dilutions of SNO-MP in triplicate were incubated with isolates in suspension for 6 hours followed by plating on tryptic soy agar and overnight incubation followed by CFU quantification. Statistical analysis was performed with SPSS using one-way analysis of variance with Bonferroni correction.

 

Results: SNO-MP displayed antibacterial activity against gram-positive (methicillin-resistant and -sensitive Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa, Enterobacter aerogenes, and Proteus mirabilis) isolates. SNO-MP induced dose-dependent reductions in CFU across all strains. Compared with controls and blank nanoparticles, SNO-MP (10 mg/mL) induced a 99.99%-100% reduction in CFU across all isolates, equivalent to a 5–9 log kill (p < 0.005). There was no statistically significant difference in CFU concentration between controls and blank microparticles. Conclusion: SNO-MP demonstrates potent bactericidal effect against antibiotic-resistant CRS bacterial strains.

Topical nitric oxide releasing nanoparticles are effective in a murine model of dermal Trichophyton rubrum dermatophytosis

Abstract

Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects and limited tissue penetration, and antimicrobial resistance is a growing concern.

 

To address this, we investigated topical nitric oxide-releasing nanoparticles (NO-np), which have been used against superficial fungal infections and bacterial abscesses. In addition to enhanced penetration and permeation conferred by nanoparticles, nitric oxide, a broad-spectrum multi-mechanistic antimicrobial agent, offers decreased likelihood of resistance development. In the current study, NO-np inhibited Trichophyton rubrum in vitro, as well as in a murine model of dermal dermatophytosis. In mice, NO-np reduced fungal burden after three days, with complete clearance after seven.

 

Furthermore, NO-np decreased tissue IL-2, 6, 10 and TNFα, indicating earlier attenuation of the host inflammatory response and decreased tissue morbidity.

Thus, topical NO-np represent an attractive alternative to systemic therapy against dermal T. rubrum infection.

Thermostable exoshells fold and stabilize recombinant proteins

Abstract

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold.

 

Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.

Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex

Abstract

Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa.

 

Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively.

 

Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex

Abstract

Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa.

 

Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively.

 

Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

Characterization and assessment of nanoencapsulated sanguinarine chloride as a potential treatment for melanoma

Abstract

Sanguinarine has a history of use in both folk medicine and early dermatology for the treatment of cutaneous neoplasms. Applied indiscriminately, bloodroot is an escharotic agent with potential to cause extensive tissue necrosis. However, when used in a controlled fashion, sanguinarine imparts selective cytotoxic/anti-proliferative activity through multiple mechanisms against human/ murine melanoma. To exploit sanguinarine's observed activity against melanoma, a targeted delivery system is required. We present a sol-gel based nanoparticulate platform for encapsulating sanguinarine chloride(sang-np)-a targeted therapeutic capable of steady, reliable delivery of predictable quantities of drug over a sustained time period with minimal undesirable effects.

 

Size and release kinetics of sang-np were characterized using dynamic light scattering and ultraviolet-visible spectroscopy respectively. In vitro efficacy of sang-np was assessed. At both 2 and 24 hours, free sanguinarine killed > 90% of B16 melanoma cells, assessed via MTT assay. At 2 hours, sang-np killed a portion of melanoma cells, increasing to percentages comparable to free sanguinarine by 24 hours. Control(empty) nanoparticles exerted minimal toxicity to melanoma cells at both time points.

 

TUNEL assay revealed that treatment with both sanguinarine and sang-np induces apoptosis in B16 melanoma cells, suggesting that both treatments act via the same mechanism of action. These data confirm controlled release of sanguinarine from sang-np, as well as comparable efficacy and mechanism of action to sanguinarine alone. This suggests that nanoparticle delivery of sanguinarine may be a unique approach to capitalize on this potent agent's inherent anti-tumor activity and overcome many of the limitations with its current formulation.

Characterization and assessment of nanoencapsulated sanguinarine chloride as a potential treatment for melanoma

Abstract

Sanguinarine has a history of use in both folk medicine and early dermatology for the treatment of cutaneous neoplasms. Applied indiscriminately, bloodroot is an escharotic agent with potential to cause extensive tissue necrosis. However, when used in a controlled fashion, sanguinarine imparts selective cytotoxic/anti-proliferative activity through multiple mechanisms against human/ murine melanoma. To exploit sanguinarine's observed activity against melanoma, a targeted delivery system is required. We present a sol-gel based nanoparticulate platform for encapsulating sanguinarine chloride(sang-np)-a targeted therapeutic capable of steady, reliable delivery of predictable quantities of drug over a sustained time period with minimal undesirable effects.

 

Size and release kinetics of sang-np were characterized using dynamic light scattering and ultraviolet-visible spectroscopy respectively. In vitro efficacy of sang-np was assessed. At both 2 and 24 hours, free sanguinarine killed > 90% of B16 melanoma cells, assessed via MTT assay. At 2 hours, sang-np killed a portion of melanoma cells, increasing to percentages comparable to free sanguinarine by 24 hours. Control(empty) nanoparticles exerted minimal toxicity to melanoma cells at both time points.

 

TUNEL assay revealed that treatment with both sanguinarine and sang-np induces apoptosis in B16 melanoma cells, suggesting that both treatments act via the same mechanism of action. These data confirm controlled release of sanguinarine from sang-np, as well as comparable efficacy and mechanism of action to sanguinarine alone. This suggests that nanoparticle delivery of sanguinarine may be a unique approach to capitalize on this potent agent's inherent anti-tumor activity and overcome many of the limitations with its current formulation.

Curcumin-encapsulated nanoparticles as innovative antimicrobial and wound healing agent

Abstract

Burn wounds are often complicated by bacterial infection, contributing to morbidity and mortality. Agents commonly used to treat burn wound infection are limited by toxicity, incomplete microbial coverage, inadequate penetration, and rising resistance. Curcumin is a naturally derived substance with innate antimicrobial and wound healing properties.

 

Acting by multiple mechanisms, curcumin is less likely than current antibiotics to select for resistant bacteria. Curcumin's poor aqueous solubility and rapid degradation profile hinder usage; nanoparticle encapsulation overcomes this pitfall and enables extended topical delivery of curcumin. In this study, we synthesized and characterized curcumin nanoparticles (curc-np), which inhibited in vitro growth of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa in dose-dependent fashion, and inhibited MRSA growth and enhanced wound healing in an in vivo murine wound model. Curc-np may represent a novel topical antimicrobial and wound healing adjuvant for infected burn wounds and other cutaneous injuries.

 

From the Clinical Editor: This team of authors created curcumin nanoparticles in an effort to overcome curcumin's endogenously poor aqueous solubility and demonstrated in a murine model its antimicrobial and wound healing properties that also resulted in growth inhibition of MRSA and Pseudomonas aeruginosa.

Imprisoned lightning: charge transport in trehalose-derived sugar glasses

Abstract

Trehalose is a naturally occurring disaccharide noted for its ability to preserve the biological function of proteins and cell membranes during periods of stress—such as water deprivation or extreme temperature—by stabilizing the conformations of the macromolecules within a glassy matrix. This phenomenon makes use of the propensity for trehalose to interact strongly with protein functional groups and solvent water molecules via hydrogen bonding.

 

Previously, it has been shown that trehalose sugar glasses also support long-range charge transport in oxidation-reduction reactions occurring between spatially separated donors and acceptors. Here, through the use of bulk Arrhenius DC-conductivity measurements, we infer that this anomalously high carrier mobility is due to proton hopping along a hydrogen bonding network formed by sorbed “water wires,” a process known as the Grotthuss mechanism.

 

Additionally, we find that the apparent activation energy of the conductivity depends non-monotonically on the bias voltage. The possibility is raised for novel photovoltaic devices based on the entrapment of photosynthetic proteins within these glasses.

Localized increase of tissue oxygen tension by magnetic targeted drug delivery

Abstract

Hypoxia is the major hindrance to successful radiation therapy of tumors. Attempts to increase the oxygen (O2) tension (PO2) of tissue by delivering more O2 have been clinically disappointing, largely due to the way O2 is transported and released by the hemoglobin (Hb) within the red blood cells (RBCs). Systemic manipulation of O2 transport increases vascular resistance due to metabolic autoregulation of blood flow to prevent over oxygenation. This study investigates a new technology to increase O2 delivery to a target tissue by decreasing the Hb-O2 affinity of the blood circulating within the targeted tissue. As the Hb-O2 affinity decreases, the tissue PO2 to satisfy tissue O2 metabolic needs increases without increasing O2 delivery or extraction.

 

Paramagnetic nanoparticles (PMNPs), synthetized using gadolinium oxide, were coated with the cell permeable Hb allosteric effector L35 (3,5-trichlorophenylureido-phenoxy- methylpropionic acid). L35 decreases Hb affinity for O2 and favors the release of O2. The L35-coated PMNPs (L35-PMNPs) were intravenously infused (10 mg kg-1) to hamsters instrumented with the dorsal window chamber model.

 

A magnetic field of 3 mT was applied to localize the effects of the L35-PMNPs to the window chamber. Systemic O2 transport characteristics and microvascular tissue oxygenation were measured after administration of L35-PMNPs with and without magnetic field. The tissue PO2 in untreated control animals was 25.2 mmHg. L35-PMNPs without magnetic field decreased tissue PO2 to 23.4 mmHg, increased blood pressure, and reduced blood flow, largely due to systemic modification of Hb-O2 affinity. L35-PMNPs with magnetic field increased tissue PO2 to 27.9 mmHg, without systemic or microhemodynamic changes. These results indicate that localized modification of Hb-O2 affinity can increase PO2 of target tissue without affecting systemic O 2 delivery or triggering O2 autoregulation mechanisms. This technology can be used to treat local hypoxia and to increase O2 in tumors, enhancing the efficacy of radiation therapies.

Topically applied no-releasing nanoparticles can increase intracorporal pressure and elicit spontaneous erections in a rat model of radical prostatectomy

Abstract

Introduction: Patients undergoing radical prostatectomy (RP) suffer from erectile dysfunction (ED) refractory to phosphodiesterase 5 inhibitors, which act downstream of cavernous nerve (CN)-mediated release of nitric oxide (NO). Direct delivery of NO to the penis could potentially circumvent this limitation. Aim: This study aimed to determine if topically applied NO-releasing nanoparticles (NO-NPs) could elicit erections in a rat model of RP through increased blood flow. Methods: Twenty-six Sprague Dawley rats underwent bilateral transection of the CN. One week later, NO-NPs were applied topically to the penile shaft in dimethylsulfoxide (DMSO) gel (10 animals) or coconut oil (6 animals). Control animals were treated with empty NPs. Erectile function was determined through the intracorporal pressure/blood pressure ratio (ICP/BP). The effect of the NO-NPs on blood flow was determined using a hamster dorsal window chamber. Main Outcome Measures: Animals were investigated for spontaneous erections, onset and duration of erectile response, and basal ICP/BP ratio. Microcirculatory blood flow was determined through measurements of arteriolar and venular diameter and red blood cell velocity.

 

Results: Eight of 10 animals treated with NO-NPs suspended in DMSO gel had significant increases in basal ICP/BP, and 6 out of these 10 animals demonstrated spontaneous erections of approximately 1 minute in duration. Time to onset of spontaneous erections ranged from 5 to 37 minutes, and they occurred for at least 45 minutes. Similar results were observed with NO-NPs applied in coconut oil. No erectile response was observed in control animal models treated with empty NPs.

 

The hamster dorsal window chamber experiment demonstrated that NO-NPs applied as a suspension in coconut oil caused a significant increase in the microcirculatory blood flow, sustained over 90 minutes. Conclusions: Topically applied NO-NPs induced spontaneous erections and increased basal ICP in an animal model of RP. These effects are most likely due to increased microcirculatory blood flow. These characteristics suggest that NO-NPs would be useful in penile rehabilitation of patients following.

Identification and structural characterization of a new three-finger toxin hemachatoxin from Hemachatus haemachatus venom.

Abstract

Snake venoms are rich sources of biologically active proteins and polypeptides. Three-finger toxins are non-enzymatic proteins present in elapid (cobras, kraits, mambas and sea snakes) and colubrid venoms. These proteins contain four conserved disulfide bonds in the core to maintain the three-finger folds.

 

Although all three-finger toxins have similar fold, their biological activities are different. A new three-finger toxin (hemachatoxin) was isolated from Hemachatus haemachatus (Ringhals cobra) venom. Its amino acid sequence was elucidated, and crystal structure was determined at 2.43 Å resolution. The overall fold is similar to other three-finger toxins. The structure and sequence analysis revealed that the fold is maintained by four highly conserved disulfide bonds. It exhibited highest similarity to particularly P-type cardiotoxins that are known to associate and perturb the membrane surface with their lipid binding sites.

 

Also, the increased B value of hemachotoxin loop II suggests that loop II is flexible and may remain flexible until its interaction with membrane phospholipids. Based on the analysis, we predict hemachatoxin to be cardiotoxic/cytotoxic and our future experiments will be directed to characterize the activity of hemachatoxin.

Glass matrix-facilitated thermal reduction: A tool for probing reactions of met hemoglobin with nitrite and nitric oxide

Abstract

Isolating elemental steps that comprise a protein reaction in solution is a difficult process. In this study, the use of sugar-derived glass matrices is evaluated as a biophysical tool to help dissect out elemental steps and isolate intermediates. Two features of the glass are utilized in this endeavor: (i) the capacity of trehalose glass matrices to support thermal reduction over macroscopic distances; and (ii) the ability of glass matrices to significantly damp large amplitude protein dynamics. The focus of the study is on the reaction of nitric oxide (NO) with a nitrite ion coordinated to the heme iron of hemoglobin (Hb).

 

The thermal reduction property of the glass is used to generate NO from nitrite within the glass, and the damping of protein dynamics is used to control entry of NO into the distal heme pocket of Hb, where it can either interact with bound nitrite or bind to the heme iron. The results not only relate to earlier controversial studies addressing the reactions of Hb with NO and nitrite but also raise the prospect that these properties of sugar-derived glassy matrices can be exploited as a new biophysical tool to modulate and probe reactions of NO with hemeproteins as well as a wide range of other metalloproteins.

Reactivity of glass-embedded met hemoglobin derivatives toward external NO: Implications for nitrite-mediated production of bioactive NO

Abstract

Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms.

 

This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties.

 

The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-dependent catalytic process whereby Hb generates relatively longlived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.

Sustained release nitric oxide releasing nanoparticles: Characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites

Abstract

A new platform using biocompatible materials is presented for generating powders comprised of nanoparticles that release therapeutic levels of nitric oxide (NO) in a controlled and sustained manner. The capacity of these particles to retain and gradually release NO arises from their having combined features of both glassy matrices and hydrogels.

 

This feature allows both for the generation of NO through the thermal reduction of added nitrite by glucose and for the retention of the generated NO within the dry particles. Exposure of these robust biocompatible nanoparticles to moisture initiates the sustained release of the trapped NO over extended time periods as determined both fluorimetrically and amperometrically.

 

The slow sustained release is in contrast to the much faster release pattern associated with the hydration-initialed NO release in powders derived from glassy matrices. These glasses are prepared using trehalose and sucrose doped with either glucose or tagatose as the source of thermal electrons needed to convert nitrite to gNO. Significantly, the release profiles for the NO in the hydrogel/glass composite materials are found to be an easily tuned parameter that is modulated through the specific additives used in preparing the hydrogel/glass composites.

 

The presented data raise the prospect that these new NO releasing nanoparticles can be easily formulated for use under a wide range of therapeutic circumstances.

Sugar-derived glasses support thermal and photo-initiated electron transfer processes over macroscopic distances

Abstract

Trehalose-derived glasses are shown to support long range electron transfer reactions between spatially well separated donors and protein acceptors. The results indicate that these matrices can be used not only to greatly stabilize protein structures but also to facilitate both thermal and photo-initiated hemeprotein reduction over large macroscopic distances.

 

To date the promise of exciting new protein-based technologies that can harness the exceptional tunability of protein functionality has been significantly thwarted by both intrinsic instability and stringent solvent/environment requirements for the expression of functional properties.

 

The presented results raise the prospect of overcoming these limitations with respect to incorporating redox active proteins into solid state devices such as tunable batteries, switches, and solar cells.

 

The findings also have implications for formulations intended to enhance long term storage of biomaterials, new protein-based synthetic strategies, and biophysical studies of functional intermediates trapped under nonequilibrium conditions. In addition, the study shows that certain sugars such as glucose or tagatose, when added to redox-inactive glassy matrices, can be used as a source of thermal electrons that can be harvested by suitable redox active proteins, raising the prospect of using common sugars as an electron source in solid state thermal fuel cells.

Probing solvation-shell hydrogen binding in glassy and sol-gel matrixes through vibronic sideband luminescence spectroscopy

Abstract

The luminescence spectrum from Gd(III) contains vibronic sidebands that provide the vibrational frequencies of molecules that comprise the solvation shell surrounding this cation. The vibronic sidebands obtained from Gd(III) in porous sol-gel matrixes as a function of added glycerol and in trehalose-derived glassy matrixes are used to probe matrix- and solvent-induced changes in the hydrogen bonding between first- and second-shell solvent molecules.

 

The results provide direct insight into the origin of the damped dynamics observed for guest proteins within these types of matrixes. The results are also consistents with the retention of water within the first solvation shell for both the glassy matrixes and the glycerol-bathed sol-gel.

Binding and release of iron by gel-encapsulated human transferrin: Evidence for a conformational search

Abstract

Human transferrin is a single-chain bilobal protein with each of the two similar but not identical lobes in turn composed of two domains. Each lobe may assume one of two stable structural conformations, open or closed, determined by a rigid rotation of the domains with respect to each other. In solution, the transformation of a lobe between open and closed conformations is associated with the release or binding of an Fe(III) ion.

 

The results of the present study indicate that encapsulation of transferrin within a porous sol-gel matrix allows for a dramatic expansion, to days or weeks, of this interconversion time period, thus providing an opportunity to probe heretofore inaccessible transient intermediates. Sol-gel-encapsulated iron-free transferrin samples are prepared by using two protocols. In the first protocol, the equilibrium form of apotransferrin is encapsulated in the sol-gel matrix, whereas in the second protocol holotransferrin is first encapsulated and then iron is removed from the protein.

 

Results of kinetic and spectroscopic studies allow for distinguishing between two models for iron binding. In the first, iron is assumed to bind to amino acid ligands of one domain, inducing a rigid rotation of the second domain to effect closure of the interdomain cleft. In the second, iron undertakes a conformational search among the thermally accessible states of the lobe, "choosing" the state which most nearly approximates the stable closed state when iron is bound. Our experimental results support the second mechanism.

Unfolding and refolding of Sol - Gel encapsulated carbonmonoxymyoglobin: An orchestrated spectroscopic study of intermediates and kinetics

Abstract

The pH-induced unfolding and refolding of the carbon monoxide-bound derivative of horse skeletal myoglobin (COMb) encapsulated in porous sol-gels is probed using several optical techniques in conjunction with different unfolding/refolding protocols. UV resonance Raman (UVRR) spectroscopy and fluorescence are used to monitor the unfolding of the globin and exposure of the A helix to solvent.

 

Absorption spectra, visible resonance Raman (VRR) spectra, and geminate recombination are used to probe the heme and the heme environment. Encapsulation slows the kinetics of acid-induced unfolding and dramatically slows the kinetics of refolding. The spectra and the kinetics imply that this approach allows for the detailed study of the burst phase of unfolding.

 

Using different encapsulation protocols and sequences of solvent replacements, it is possible to trap and probe not only low-pH forms observed in solution-phase studies but also novel partially unfolded species that are likely to be important unfolding and folding intermediates. The role of water as a chaotropic agent is indicated by the spectral changes that occur in the introduction and subsequent removal of glycerol from the solution bathing the unfolded and partially unfolded, sol - gel encapsulated COMb.

 

The results directly support the view that unfolding or increasing the exposure to solvent of at least some segment of the A helix is the initial step in the unfolding pathway. In addition, the results indicate that the refolding of the A helix is likely to be the last process in the refolding pathway.