The Influence of Chemical Descriptors on the Reactivity of Potential Hypergolic Fuels With Hydrogen Peroxide
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Handling Procedures of Vegetable Crops
The National Aeronautics and Space Administration (NASA) is working towards future long duration manned space flights beyond low earth orbit. The duration of these missions may be as long as 2.5 years and will likely include a stay on a lunar or planetary surface. The primary goal of the Advanced Food System in these long duration exploratory missions is to provide the crew with a palatable, nutritious, and safe food system while minimizing volume, mass, and waste. Vegetable crops can provide the crew with added nutrition and variety. These crops do not require any cooking or food processing prior to consumption. The vegetable crops, unlike prepackaged foods, will provide bright colors, textures (crispy), and fresh aromas. Ten vegetable crops have been identified for possible use in long duration missions. They are lettuce, spinach, carrot, tomato, green onion, radish, bell pepper, strawberries, fresh herbs, and cabbage. Whether these crops are grown on a transit vehicle (e.g., International Space Station) or on the lunar or planetary surface, it will be necessary to determine how to safely handle the vegetables while maintaining acceptability. Since hydrogen peroxide degrades into water and oxygen and is generally recognized as safe (GRAS), hydrogen peroxide has been recommended as the sanitizer. The objective of th is research is to determine the required effective concentration of hydrogen peroxide. In addition, it will be determined whether the use of hydrogen peroxide, although a viable sanitizer, adversely affects the quality of the vegetables. Vegetables will be dipped in 1 % hydrogen peroxide, 3% hydrogen peroxide, or 5% hydrogen peroxide. Treated produce and controls will be stored in plastic bags at 5 C for up to 14 days. Sensory, color, texture, and total plate count will be measured. The effect on several vegetables including lettuce, radish, tomato and strawberries has been completed. Although each vegetable reacts to hydrogen peroxide differently, the data suggest that 5% hydrogen peroxide reduces the shelf life of the vegetable. A dip of either 1 % or 3% hydrogen peroxide helps reduce the microbial total count while not adversely affecting the quality of the vegetable.
Perchonok, Michele↗
On copper peroxide
The action of hydrogen superoxide on copper salts in alcoholic solutions is studied. The action of hydrogen peroxide on copper hydroxide in alcoholic suspensions, and the action of ethereal hydrogen peroxide on copper hydroxide are discussed. It is concluded that using the procedure proposed excludes almost entirely the harmful effect of hydrolysis.
Moser, L.↗
Efficacy of Various Chemical Disinfectants on Biofilms Formed in Spacecraft Potable Water System Component
As the provision of potable water is critical for successful habitation of the International Space Station (ISS), life support systems were installed in December 2008 to recycle both humidity from the atmosphere and urine to conserve available water in the vehicle. Pre-consumption testing from the dispensing needle at the Potable Water Dispenser (PWD) indicated that bacterial concentrations exceeded the current ISS specifications of 50 colony forming units (CFU) per ml. Subsequent investigations revealed that a corrugated stainless steel flex hose upstream of the dispensing needle in the PWD was filled with non-sterile water and left at room temperature for over one month before launch. To simulate biofilm formation that was suspected in the flight system, sterile flex hoses were seeded with a consortium of bacterial isolates previously recovered from other ISS water systems, which included Ralstonia pickettii, Burkholderia multivorans, Caulobacter vibrioides., and Cupriavidus pauculus. After 5 days of incubation, these hoses were challenged with various chemical disinfectants including hydrogen peroxide, colloidal silver, and buffered pH solutions to determine the ability of the disinfectants to decrease and maintain bacterial concentrations below ISS specifications. Disinfection efficacy over time was measured by collecting daily heterotrophic plate counts following exposure to the disinfectants. A single flush with either 6% hydrogen peroxide solution or a mixture of 3% hydrogen peroxide and 400 ppb colloidal silver effectively reduced the bacterial concentrations to less than 1 CFU/ml for a period of up to 2 months. Testing results indicated that hydrogen peroxide and mixtures of hydrogen peroxide and colloidal silver have tremendous potential as alternative disinfectants for ISS water systems.
Wong, Willy↗
Organometallic catalysts for primary phosphoric acid fuel cells
A continuing effort by the U.S. Department of Energy to improve the competitiveness of the phosphoric acid fuel cell by improving cell performance and/or reducing cell cost is discussed. Cathode improvement, both in performance and cost, available through the use of a class of organometallic cathode catalysts, the tetraazaannulenes (TAAs), was investigated. A new mixed catalyst was identified which provides improved cathode performance without the need for the use of a noble metal. This mixed catalyst was tested under load for 1000 hr. in full cell at 160 to 200 C in phosphoric acid H3PO4, and was shown to provide stable performance. The mixed catalyst contains an organometallic to catalyze electroreduction of oxygen to hydrogen peroxide and a metal to catalyze further electroreduction of the hydrogen peroxide to water. Cathodes containing an exemplar mixed catalyst (e.g., Co bisphenyl TAA/Mn) operate at approximately 650 mV vs DHE in 160 C, 85% H3PO4 with oxygen as reactant. In developing this mixed catalyst, a broad spectrum of TAAs were prepared, tested in half-cell and in a rotating ring-disk electrode system. TAAs found to facilitate the production of hydrogen peroxide in electroreduction were shown to be preferred TAAs for use in the mixed catalyst. Manganese (Mn) was identified as a preferred metal because it is capable of catalyzing hydrogen peroxide electroreduction, is lower in cost and is of less strategic importance than platinum, the cathode catalyst normally used in the fuel cell.
Walsh, Fraser↗
Hydrogen Peroxide-Water-Ethanol-Monopropellant Blend for CubeSat Propulsion
Monopropellant propulsion that provides high performance and low hazards compared to state-of-art hydrazine are being investigated under NASA, DOD and commercially sponsored green propellant research and development programs. This NASAeffort looks at phosphate stabilized 50% hydrogen peroxide/water –ethanol blends to evaluate their stability and combustion behavior in submillimeter diameter microtube combustion apparatus for cubesat propulsion applications. Exploratory 7-day stabilitytests find no pressure rise in a sealed test tube environment at room temperature indicating at least short term stability of the mixture. Temperature rise during combustion testing in resistively heated nickel microtube apparatus shows that micro-combustion is achieved in the tube. Propellant density is approximately 1.2 g/cm3compared to 1.0 g/cm3for hydrazine. Theoretical specific impulse of the stoichiometric blend is 219 s at a chamber pressure of 20 psia and an area ratio of 30:1. Density-specific impulseof this blendis263 swhile hydrazine is only about 215-220 sin small reaction control system thrusters. This provides about 20-22% more density-specific impulse for the cubesat compared to hydrazine. The theoreticalcombustion temperature is2168°F, whichis thermally compatible with stainless steel materials of constructionand the nickel catalystused in the tests. Combustion test results are given for stoichiometric, as well as fuel-leanand fuel-richpropellant formulations. Thesetests focus on thermally and catalytically supported combustionin the microtube, whichrequires a microtube preheat temperature of about 900°F and sufficient residence time to achieve combustion that is stable and anchored in the tubewithout blowing through the tube unreacted. Thrust class is expected to be in the 10 mN range based on flow rate and estimated specific impulse.Power requirements are generally below 25 Wto heat the tube. Steady state combustion testing is typically conducted for 3 minutes and shows littledegradation of the hardware over multiple test cyclesindicating thatthere is little catalyst degradation over time and littlecatalyst deactivation due tothe phosphate stabilizer inthe hydrogen peroxide.
Monopropellant↗
Hydrogen Peroxide-Water-Ethanol Monopropellant Blend for CubeSat Propulsion
Monopropellant propulsion that provides high performance and low hazards compared to state-of-art hydrazine are being investigated under NASA, DOD and commercially sponsored green propellant research and development programs. This NASA effort looks at phosphate stabilized 50% hydrogen peroxide/water –ethanol blends to evaluate their stability and combustion behavior in submillimeter diameter microtube combustion apparatus for cubesat propulsion applications. Exploratory 7-day stability tests find no pressure rise in a sealed test tube environment at room temperature indicating at least short term stability of the mixture. Temperature rise during combustion testing in resistively heated nickel microtube apparatus shows that micro-combustion is achieved in the tube. Propellant density is approximately 1.2 g/cu.cm compared to 1.0 g/cu.cm for hydrazine. Theoretical specific impulse of the stoichiometric blend is 219 s at a chamber pressure of 20 psia and an area ratio of 30:1. Density-specific impulse of this blend is 263 s while hydrazine is only about 215-220 s in small reaction control system thrusters. This provides about 20-22% more density-specific impulse for the cube sat compared to hydrazine. The the oretical combustion temperature is 2168°F, which is thermally compatible with stainless steel materials of construction and the nickel catalyst used in the tests. Combustion test results are given for stoichiometric, as well as fuel-lean and fuel-rich propellant formulations. These tests focus on thermally and catalytically supported combustion in the microtube, which requires a microtube preheat temperature of about 900°F and sufficient residence time to achieve combustion that is stable and anchored in the tube without blowing through the tube unreacted. Thrust class is expected to be in the 10 m N range based on flow rate and estimated specific impulse. Power requirements are generally below 25 W to heat the tube. Steady state combustion testing is typically conducted for 3 minutes and shows little degradation of the hardware over multiple test cycles indicating that there is little catalyst degradation over time and little catalyst deactivation due to the phosphate stabilizer in the hydrogen peroxide.
Monopropellant↗
Development of biological and nonbiological explanations for the Viking label release data
The plausibility that hydrogen peroxide, widely distributed within the Mars surface material, was responsible for the evocative response obtained by the Viking Labeled Release (LR) experiment on Mars was investigated. Although a mixture of gamma Fe2O3 and silica sand stimulated the LR nutrient reaction with hydrogen peroxide and reduced the rate of hydrogen decomposition under various storage conditions, the Mars analog soil prepared by the Viking Inorganic Analysis Team to match the Mars analytical data does not cause such effects. Nor is adequate resistance to UV irradiation shown. On the basis of the results and consideration presented while the hydrogen peroxide theory remains the most, if not only, attractive chemical explanation of the LR data, it remains unconvincing on critical points. Until problems concerning the formation and stabilization of hydrogen peroxide on the surface of Mars can be overcome, adhere to the scientific evidence requires serious consideration of the biological theory.
Source record↗
A METHOD TO REDUCE BIOBURDEN IN ASTROMATERIALS CURATION FACILITIES WITHOUT INTRODUCING UNWANTED CONTAMINATION
Introduction: NASA curates its Astromaterials collections in cleanrooms that are carefully monitored for particulate, inorganic and trace metal contamination. Current sample collections are not particularly susceptible to organic contamination or biological alteration. However, new collections like those from the OSIRIS-REx and Hayabusa2 missions will have organic contamination requirements and are susceptible to biodegradation. It will be necessary sterilize or at least disinfect curation labs, as well as tools and equipment in a manner that does not introduce additional contamination and does not affect the samples 1. Current curation cleaning procedures utilize isopropyl alcohol which offers some bioburden reduction, but is not effective against spore-forming bacteria or fungal spores 2. We present a modified disinfection method that uses ultrapure hydrogen peroxide to reduce bioburden inside curation labs and glove boxes without introducing contamination or damaging curation equipment. We tested this method in the meteorite processing lab as well as on a glovebox being cleaned for use in processing ANGSA (Apollo Next Generation Sample Analysis) samples and present the results of those tests. We discuss the limitations of this method and describe potential situations in which it will not be applicable. The CDC guidelines for disinfection andsterilization in healthcare facilities discusses over 15different methods for reducing bioburden in hospitalsettings 3. The most common method, steamsterilization, is well suited to sterilizing curationprocessing tools but cannot easily be used to sterilizecleanroom surfaces or large equipment likegloveboxes. Chemical sterilization with bleach(NaOCl) is also a common strategy in healthcare andpharmaceutical settings that presents materialcompatibility issues as well as serious inorganiccontamination concerns for curation facilities.Introducing a new source of Na and Cl into curationlabs is not acceptable. Other chemical methods likeethylene oxide, formaldehyde, iodophors andquaternary ammonium compounds could introduceorganic and inorganic contamination. We chose tofocus on hydrogen peroxide because it is generallycompatible with commonly used curation materialslike stainless steel, aluminum and Teflon and becauseit decomposes to oxygen and water. The CDCguidelines for hydrogen peroxide specify using a 7.5wt% solution at 25 ̊C with a contact time of 30 minutesfor high level disinfection and 6 hours for sterilization.High level disinfection is defined as a technique thatwill kill all microorganisms except large numbers ofbacterial spores 3. Methods: We prepared a solution of 7.5 wt%hydrogen peroxide from a stock solution of ultrapure30 wt% peroxide (JT Baker) and curation gradeultrapure water. This ultrapure water is already used incuration cleaning procedures and thus is not consideredand additional source of contamination. We conducteda materials compatibility test by exposing unanodizedand anodized 6061 T6 Al alloy to the peroxide solutionfor up to six hours and periodically inspecting thesurfaces for visible defects. We used this peroxide todisinfect the floor of the meteorite processing lab andthe interior of a curation glovebox by exposing thesesurfaces to the peroxide solution for 30 min. Thesurfaces were swabbed with a dry macrofoam swabbefore (Puritan Brand 2518051PFRNDFD) and afterperoxide treatment to collect microbes present on thesurfaces. Microbes were extracted by sonication fromthe swab into 15 ml of PBS (phosphate buffered saline)and inoculated onto the following media: TSA (trypticsoy agar) BA (blood agar), R2A (Reasoners 2 agar),Potato Dextrose Agar, Saboraud Dextrose Agar andSaboraud Dextrose Agar with 0.1 mg/ mlchloramphenicol. Four TSA plates and two BA plateswere inoculated with 0.1 ml of PBS each andincubated at 35 and 37 for 48 hours. Two R2A°C°Cplates (0.1 ml of PBS each) were incubated at 25 .°CThe remaining plates were inoculated with 0.2ml ofPBS and incubated at 30 ̊C for seven days. Afterincubation bacterial and fungal isolates were countedand transferred to new plates for identification usingthe VITEK24 automated system or by sequencing aportion of the barcode gene (16S rRNA for bacteria,small subunit gene for fungi) on an ABI 3500 Sangersequencer. Negative controls consisted of swabs thatwere opened in the sampling environment andanalyzed alongside the experimental samples.Results: A 6 hour exposure to hydrogen peroxideresulted in visible pitting on un-anodized 6061 Al, butnot on anodized surfaces. No visible pitting occurredafter a 30 minute exposure. Therefore, we decided tolimit our experimental tests to 30 min. exposures. 17bacterial CFU (colony forming units) representing 4distinct organisms were isolated from the meteorite processing lab floor prior to hydrogen peroxidetreatment. We were unable culture any organisms afterperoxide treatment. In the glovebox we were able toculture three bacterial CFU representing three distinctspecies, including a spore forming bacterium prior todisinfection with peroxide. After the peroxidetreatment we were unable to culture any organisms.Routine monitoring of the meteorite processing lab andthe glovebox did not indicate any increase in unwantedinorganic contamination after these peroxidetreatments. Discussion: A 30 minute treatment with 7.5 wt%peroxide appears to be an effective method forreducing bioburden on typical cleanroom surfaces. Themethod does not introduce unwanted organic orinorganic contamination and is compatible withcommonly used curation materials like stainless steel,Teflon and anodized aluminum alloys. Special careshould be taken with un-anodized aluminum.Prolonged exposure to hydrogen peroxide can causepitting on this material. We recommend using thismethod to disinfect curation labs and equipment whenbiological alteration is a concern. This method iseffective at room temperature and cannot be used todisinfect labs and equipment where the ambienttemperature is < 0 ̊C. Astromaterials samples shouldbe removed from the area where disinfection is tooccur. Hydrogen peroxide is a powerful oxidizingagent and will react with any organic carbon present inthe sample. References: [1.] Mccubbin, F. M. et al.Sp. Sci Rev(2019) doi:10.1007/s11214-019-0615-9. [2.] Mogul, R.et al.Astrobiology 18, ast.2017.1814 (2018). [3.]Rutala, W. A. & Weber, D. J. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. [4.] Pincus, D. H. in Encyclopedia of Rapid Microbiological Methods (2005).
A. B. Regberg↗
Investigation of new hypergol scrubber technology
The ultimate goal of this work is to minimize the liquid waste generated from the scrubbing of hypergolic vent gases. In particular, nitrogen tetroxide, a strong oxidizer used in hypergolic propellant systems, is currently scrubbed with a sodium hydroxide solution resulting in a hazardous liquid waste. This study investigated the use of a solution of potassium hydroxide and hydrogen peroxide for the nitrogen textroxide vent scrubber system. The potassium nitrate formed would be potentially usable as a fertilizer. The hydrogen peroxide is added to convert the potassium nitrite that is formed into more potassium nitrate. Smallscale laboratory tests were conducted to establish the stability of hydrogen peroxide in the proposed scrubbing solution and to evaluate the effectiveness of hydrogen peroxide in converting nitrite to nitrate.
Glasscock, Barbara H.↗
Composite-Material Tanks with Chemically Resistant Liners
Lightweight composite-material tanks with chemically resistant liners have been developed for storage of chemically reactive and/or unstable fluids . especially hydrogen peroxide. These tanks are similar, in some respects, to the ones described in gLightweight Composite-Material Tanks for Cryogenic Liquids h (MFS-31379), NASA Tech Briefs, Vol. 25, No. 1 (January, 2001), page 58; however, the present tanks are fabricated by a different procedure and they do not incorporate insulation that would be needed to prevent boil-off of cryogenic fluids. The manufacture of a tank of this type begins with the fabrication of a reusable multisegmented aluminum mandrel in the shape and size of the desired interior volume. One or more segments of the mandrel can be aluminum bosses that will be incorporated into the tank as end fittings. The mandrel is coated with a mold-release material. The mandrel is then heated to a temperature of about 400 F (approximately equal to 200 C) and coated with a thermoplastic liner material to the desired thickness [typically approxiamtely equal to 15 mils (approximately equal to 0.38 mm)] by thermal spraying. In the thermal-spraying process, the liner material in powder form is sprayed and heated to the melting temperature by a propane torch and the molten particles land on the mandrel. The sprayed liner and mandrel are allowed to cool, then the outer surface of the liner is chemically and/or mechanically etched to enhance bonding of a composite overwrap. The etched liner is wrapped with multiple layers of an epoxy resin reinforced with graphite fibers; the wrapping can be done either by manual application of epoxy-impregnated graphite cloth or by winding of epoxy-impregnated filaments. The entire assembly is heated in an autoclave to cure the epoxy. After the curing process, the multisegmented mandrel is disassembled and removed from inside, leaving the finished tank. If the tank is to be used for storing hydrogen peroxide, then the liner material should be fluorinated ethylene/propylene (FEP), and one or more FEP O ring(s) should be used in the aluminum end fitting(s). This choice of materials is dictated by experimental observations that pure aluminum and FEP are the only materials suitable for long-term storage of hydrogen peroxide and that other materials tend to catalyze the decomposition of hydrogen peroxide to oxygen and water. Other thermoplastic liner materials that are suitable for some applications include nylon 6 and polyethylene. The processing temperatures for nylon 6 are lower than those for FEP. Nylon 6 is compatible with propane, natural gas, and other petroleum-based fuels. Polyethylene is compatible with petroleum- based products and can be used for short-term storage of hydrogen peroxide.
DeLay, Thomas K.↗
Process and Equipment for Nitrogen Oxide Waste Conversion to Fertilizer
The present invention describes a process for converting vapor streams from sources containing at least one nitrogen-containing oxidizing agent therein to a liquid fertilizer composition comprising the steps of: (1) directing a vapor stream containing at least nitrogen-containing oxidizing agent to a first contact zone; (2) contacting said vapor stream with water to form nitrogen oxide(s) from said at least one nitrogen- containing oxidizing agent; (3) directing said acid(s) as a second stream to a second contact zone; (4) exposing said second stream to hydrogen peroxide which is present within said second contact zone in a relative amount of at least 0.1% by weight of said second stream within said second contact zone to convert at least some of any nitrogen oxide species or ions other than in the nitrite form present within said second stream to nitrate ion; (5) sampling said stream within said second contact zone to determine the relative amount of hydrogen peroxide within said second contact zone; (6) adding hydrogen peroxide to said second contact zone when a level on hydrogen peroxide less than 0.1% by weight in said second stream is determined by said sampling; (7) adding a solution comprising potassium hydroxide to said second stream to maintain a pH between 6.0 and 11.0 within said second stream within said second contact zone to form a solution of potassium nitrate; and (8) removing sais solution of potassium nitrate from said second contact zone.
Lueck, Dale E.↗
Process for Nitrogen Oxide Waste Conversion to Fertilizer
The present invention describes a process for converting vapor streams from sources containing at least one nitrogen-containing oxidizing agent therein to a liquid fertilizer composition comprising the steps of: a) directing a vapor stream containing at least one nitrogen-containing oxidizing agent to a first contact zone; b) contacting said vapor stream with water to form nitrogen oxide(s) from said at least one nitrogen-containing oxidizing agent; c) directing said acid(s) as a second stream to a second contact zone; d) exposing said second stream to hydrogen peroxide which is present within said second contact zone in a relative amount of at least 0.1% by weight of said second stream within said second contact zone to convert at least some of any nitrogen oxide species or ions other than in the nitrate form present within said second stream to nitrate ion; e) sampling said stream within said second contact zone to determine the relative amount of hydrogen peroxide within said second contact zone; f) adding hydrogen peroxide to said second contact zone when a level of hydrogen peroxide less than 0.1 % by weight in said second stream is determined by said sampling; g) adding a solution comprising potassium hydroxide to said second stream to maintain a pH between 6.0 and 11.0 within said second stream within said second contact zone to form a solution of potassium nitrate; and h) removing said solution of potassium nitrate from said second contact zone.
Lueck, Dale E.↗
A search for a nonbiological explanation of the Viking Labeled Release life detection experiment
The possibility of nonbiological reactions involving hydrogen peroxide being the source of the positive response detected by the Viking Labeled Release (LR) life detection experiment on the surface of Mars is assessed. Labeled release experiments were conducted in the LR Test Standards Module which replicates the Viking flight instrument configuration on analog Martian soils prepared to match the Viking inorganic analysis of Mars surface material to which an aqueous solution of hydrogen peroxide had been added. Getter experiments were also conducted to compare several reactions simultaneously in the presence and absence of UV radiation prior to the addition of nutrient. Hydrogen peroxide on certain analog soils is found to be capable of reproducing the kinetics and thermal information contained in the Mars data. The peroxide concentration necessary for this response, however, is shown to require a chemical stability or production rate much greater than seems likely in the Mars environment. As previous experiments have shown hydrogen peroxide to be the most likely nonbiological source of the positive LR response, it is concluded that the presence of a biological agent on Mars must not yet be ruled out.
Levin, G. V.↗
Emission control system
Methods and apparatus utilizing hydrogen peroxide are useful to reduce NOx, SOx and mercury (or other heavy metal) emissions from combustion flue gas streams. Continuous concentration of hydrogen peroxide to levels approaching or exceeding propellant-grade hydrogen peroxide facilitates increased system efficiency. In this manner, combustion flue gas streams can be treated for the removal of NOx, SOx and heavy metals, while isolating useful by-products streams of sulfuric acid and nitric acid as well as solids for the recovery of the heavy metals.
Parrish, Clyde F.↗
Emission control system
Methods and apparatus utilizing hydrogen peroxide are useful to reduce SOx and mercury (or other heavy metal) emissions from combustion flue gas streams. The methods and apparatus may further be modified to reduce NOx emissions. Continuous concentration of hydrogen peroxide to levels approaching or exceeding propellant-grade hydrogen peroxide facilitates increased system efficiency. In this manner, combustion flue gas streams can be treated for the removal of SOx and heavy metals, while isolating useful by-products streams of sulfuric acid as well as solids for the recovery of the heavy metals. Where removal of NOx emissions is included, nitric acid may also be isolated for use in fertilizer or other industrial applications.
Parrish, Clyde F.↗
UV Induced Oxidation of Nitric Oxide
Nitric oxide in a gaseous stream is converted to nitrogen dioxide using oxidizing species generated at least in part using in situ UV radiation sources. The sources of the oxidizing species include oxygen and/or hydrogen peroxide. The oxygen may be a component of the gaseous stream or added to the gaseous stream, preferably near a UV radiation source, and is converted to ozone by the UV irradiation. The hydrogen peroxide is decomposed through a combination of vaporization and UV irradiation. The hydrogen peroxide is preferably stored at stable concentration levels, i.e., approximately 50% by volume and increased in concentration in a continuous process preceding vaporization within the flow channel of the gaseous stream and in the presence of the UV radiation sources.
Parrish, Clyde, F.↗
Effects of Microheterogeneity in Hen Egg-White Lysozyme Crystallization
In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.
Thomas, B. R.↗