Catalase Crystals Research Articles

Purification, Crystallization, and Properties of Bovine Milk Catalase

Catalase of bovine milk was crystallized following n-butanol extraction, ammonium sulfate fractionation, ethanol-chloroform fractionation, diethyl-aminoethyl-Sephacel column chromatography, and Sephacryl S-300 gel filtration. About 120mg crystalline catalase was produced from 1200kg milk. The product was approximately 23,000 times as pure as milk.The crystalline catalase appeared homogeneous on disc-electrophoretic analysis. Its molecular weight was approximately 225,000 by Sephadex G-200 gel filtration. Optimum pH and temperature of the crystalline catalase were pH 8.0 and 20°C. The enzyme had a thermostable range below 40° C. Its absorption spectrum showed two bands, one at 275nm (protein) and the other from 360 to 450nm (Soret band, heme group). About 8% sodium chloride concentration reduced catalase activity by more than 6%.

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain, glucose or embedding in the presence of tannic acid

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2 12 12 1 and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 Å. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with nonembedded, negatively stained thin platelet crystals. In addition, good agreement at 20 Å resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by lowdose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3.7 Å as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 Å resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.

Electron paramagnetic resonance crystallography of bacterial catalase: g-Contour mapping method of analysis.

Single crystals of bacterial catalase from Micrococcus luteus have been examined by EPR at 77 K. X-ray perfect crystals gave a set of four prominent resonances in all three orthogonal planes which yielded eight heme direction cosine matrices to an accuracy of +/- 2 degrees as expected for the P4(2)2(1)2 space group and unit cell parameters previously determined. These matrices are related by D4 symmetry operation of the space group. There were additional weaker resonances only resolved in two or even one plane. A method of g-contour mapping was devised to solve for the orientations of hemes that give rise to these weaker resonances. Three additional sets of heme orientations, also following D4 symmetry, were determined. All of the above sites have the same principal g values, 2.0, 5.4, and 6.6. The EPR crystallographic results imply that several conformational substates may be trapped at 77 K.

Resin development for electron microscopy and an analysis of embedding at low temperature*

SUMMARYWe have tested a wide range of acrylate and methacrylate resin formulations and have concluded that embedding resins can be developed which are usable within a broad range of environmental conditions. To demonstrate this versatility, we have designed two highly‐cross‐linked resins, one polar and the other nonpolar, that are usable to temperatures from 243 to 223 K. Both of these resins formulations, which are now commercially available, show that systematic experiments can be easily done to study the effects of environmental parameters, such as water content, temperature, or resin polarity, on biological material during embedding. Using these resins and aldehyde‐fixed protein crystals, it can be shown that low temperature minimizes the loss of molecular structure to an extent that is not often obtainable with conventional methods of dehydration and embedding. Embedded crystals of aspartate aminotransferase still retained molecular order to 0·6 nm. Embedded crystals of catalase show X‐ray diffraction maxima out to 0·8 nm. When sectioned, catalase revealed stain‐limited electron and optical diffraction patterns to 2·5 nm. Nevertheless, our work clearly demonstrates that low temperature embedding procedures are superior and that versatile, general purpose resins can be designed to take advantage of this fact.

Electron probe and electron energy loss analysis in biology

Methods, applications and limitations of quantitative electron probe analysis, X-ray mapping, electron energy loss analysis and energy filtered imaging are described, with emphais on the analysis of thin (less than 200nm) cryosections. Energy dispersion electron probe analysis can measure reliably 5 to 10mM/Kg of biologically prevalent elements in 50nm diameter areas of 100 to 150nm thick cryo sections during 100–300 sec counts. The minimal detectable mass (MDM) with a conventional thermionic electron source is approximately 10 −19g Fe (100 sec count) and can be reduced to 10 −20g through the use of a field emission gun (FEG). A spatial resolution of 8.7nm is demonstrated in two-dimensional Fourier transforms of Mo X-ray maps of stained catalase crystals. Significant biological results of quantitative electron probe analysis include the measurement of total Ca released from and Mg and K taken up by the sarcoplasmic reticulum during muscle contraction, and the demonstration that mitochondria do not contribute to the physiological regulation of cytoplasmic free Ca levels in cardiac, vascular smooth and striated muscle. Electron energy loss analysis (EELS) promises a significant improvement in sensitivity for the measurement of Ca; based on statistical errors of the measurement, 250μM/Kg Ca should be measurable with EELS in 250 sec. through the Ca L-edge loss. The use of a doubly corrected magnetic sector spectrometer as a transmission electron microscope imaging filter outside the microscope vacuum is illustrated, and the resolution of the iron core (7.5nm) and surrounding organic shell of single ferritin molecules is demonstrated in, respectively, iron M and carbon K loss images.

The Observation of Frozen Hydrated Sections by CTEM and STEM

With the ultimate aim of studying biological structures close to their native state, we are attempting to prepare and examine thin frozen hydrated sections using catalase crystals and skeletal muscle as test objects. There have been a few observations under these conditions.small droplet of catalase crystals stained in 2% aqueous uranyl acetate and re-suspended in water was placed on a copper specimen holder and plunged into liquid nitrogen. Small pieces of rat femoral muscle were fixed in 2% gluter- aldehyde in 0.05 M cacodylate buffer, and frozen as above after washing in buffer with or without the addition of 1 M sucrose.Frozen sections for examination in the CTEM were cut with glass knives in a Sorvall MT2B microtome with the LTC2 cryo-attachment operating at ca. -120°C. For examination in the STEM, frozen sections were cut in a Reichert Ultracut with FC4 cryo-attachment operating with the knife and the specimen at -150°C and the chamber at -170°C. Sections that appeared transparent were manipulated onto carbon coated grids with a pre-cooled eyelash probe and then sandwiched in place with a second carbon coated grid.

Eel Rhabdovirus, EVA, EVEX and Their Infectivity to Fishes

Negatively stained EVA and EVEX with typical rhabdovirus shapes were measured using catalase crystals as the standard. Their mean sizes were 143.2×83.5 and 144.6×83.7 nm respec tively. The CPE caused by EVA and EVEX in RTG-2 cells were very similar, but they were clearly distinguishable from that of IHNV. They induced marginal hyperchromatosis, deforma of nuclei and formation of basophilic inclusions of irregular shapes in the cytoplasm of in fected cells. Finally, infected cells became round and then collapsed in pieces. By SEM observa of CPE caused by both viruses, it was found that infected cells having long filopodia became round with some shrinking. Serologically, there is close similarity between EVA and EVEX, but both are clearly different from IHNV and probably different from VHSV type II and 23-75 strain. Infectivity trials with rainbow trout fry were carried out at 10°C, 15°C and 20°C. Both EVA EVEX showed more severe pathogenicity at higher temperatures. Symptoms and histo pathological changes of fish infected with EVA and EVEX were similar to those of IHN, and included hemorrhage of skeletal muscles, intensive necrosis and hemorrhage of hematopoietictissue of the kidneys and focal necrosis of liver, spleen and panceras. The fact that EVEX showed strong pathogenicity even at 15°C makes us concerned about the introduction of a new viral disease in rainbow trout cultured in Japan.

Elastin-associated microfibrils in aorta: species differences in large animals

As part of a comparative study of elastic tissue morphology, we have adapted a method for measuring accurately, in tissue sections, differences in the diameters of the microfibrils associated with aortic elastic tissue. The method is reproducible to within ± 2%. Measurements have been made of the diameters of microfibrils associated with the internal elastic lamellae of the thoracic descending aortae of members of four species of large animals commonly used in the study of elastic tissue. Healthy mature young adult male animals have been examined so as to avoid differences associated with development and disease. Rigorous attention has been given to standardisation of fixation, processing, microscopy and measurement. A single negatively stained catalase crystal was used as reference standard for the calibration of each series of electronmicrographics. It has been found that sheep and bovine aortic microfibrils are not significantly different in diameter. However, their mean diameters are significantly and reproducibly different from those of humans and pigs (in order of increasing diameter). The mean diameters of the microfibrils of the latter two species are also significantly different from one another. Comparison of the catalase crystal with tobacco mosaic virus and with other catalase preparations revealed highly significant and reproducible differences in the crystal lattice spacing of different catalase preparations available commercially.

Some Properties of the Virions of Galinsoga Mosaic Virus.

An increasing number of plant viruses with stable isometric virions are being described, but their classification will be possible only when the biochemical and biophysical properties of these virions are better known. This paper reports additional properties of one such virus, namely Galinsoga mosaic virus (GMV), which was originally isolated from the roots of an annual weed species Galinsoga parviflora Cav. (1) in Queensland, but has a wide host range and is soil-borne (2). Virions of GMV were purified as described by Behncken (1) using the primary leaves of bean (Phaseolus vulgaris) as both propagation and assay host. Virion concentration was correlated with symptom severity; the most concentrated preparations were obtained from the leaves of the cultivars Hawkesbury Wonder, Canadian Wonder and Royal Windsor, and least from Blue Lake, Epicure, Purple King, Redland Pioneer and Westralia. With infective sap diluted to give between 5 and 100 lesions per leaf, there was a linear correlation between sap concentration and lesion num bers, suggesting that one sort of particle determined infectivity. Properties of GMV virions were assessed by the techniques used by Skotnicki et al. (3). Virion preparations had ultraviolet absorption spectra of nucleoprotein suspensions with an A260/A2soratio of 1.66, indicating an RNA content of about 22%. When negatively stained in 1% uranylacetate, the virions were isometric with a rounded profile and a diameter of 28 nm (measured against the lattice of bovine catalase crystals). The virions had a sedimentation coefficient of 117.9 ± 2.1 S20, w(3 estimates). Some preparations also contained a component that sedimented more slowly (f. 48S) and had a much smaller A260/A280 ratio than the virions. GMV genome RNA, compared electrophoretically with 23S and 16S ribosomal RNA of E. coli in 0.5, 1.5 and 3.0% polyacrylamide gels, had an estimated molecular weight of 1.55 x 106. This RNA had a molar base composition (5 analyses) of G 24.8 ± 0.3; A 28.1 ± 0.6; C 21.8 ± 1.0; U 25.3 ± 0.7. GMV virion protein migrated as a single species in PAGE experiments and had a mean molecular weight (3 estimates) of 36.4 (± 1.8) x 10 • The percentage molar composition and 'best fitting' integral number of amino acids were assessed from samples hydrolysed for 1, 2 and 4 days and others oxidised with performic acid. The resulting estimates were Ala 7.07 (26), Arg 4.44 (16), Asx 9.30 (34), Cys 1.61 (6), Glx 8.08 (29), Gly 9.97 (36), His 2.14 (8), lie

Design and operation of a differentially pumped environmental chamber for the HVEM

A differentially pumped environmental chamber, or DIFPEC, has been developed for the AEI EM7 1.2 MV HVEM. The chamber is suitable for imaging and diffraction studies and is capable of operation at pressures approaching one atmosphere. Any mixture of gases can be used including water vapor supplied from an internal reservoir. Correct operation is demonstrated by measurements of temperature, pressure, and water consumption. High resolution (0.2 nm) diffraction patterns were recorded from unfixed, unstained, fully hydrated catalase crystals. This is good functional test of DIFPEC operation and specimen preparation because catalase disorders irreversibly of exposed to less than 95% relative humidity. High- and low-angle diffraction patterns were recorded from unfixed, unstained, fully hydrated rat hemoglobin crystals. All patterns were recorded using 10-4 C/cm2. Whole cell mounts prepared in different ways were imaged and show that 5.3 kPa of nitrogen gas has no detrimental effect on image contrast or resolution. The column vacuum and differentially pumped volume pressure were measured for a number of pumping configuration, as well as for several gases and pressures in the specimen volume. The pressure in the high vacuum portion of the objective lens gap is conductance-limited when the DIFPEC is in place.

Design and operation of a differentially pumped environmental chamber for the HVEM

A differentially pumped environmental chamber, or DIFPEC, has been developed for the AEI EM7 1.2 MV HVEM. The chamber is suitable for imaging and diffraction studies and is capable of operation at pressures approaching one atmosphere. Any mixture of gases can be used including water vapor supplied from an internal reservoir. Correct operation is demonstrated by measurements of temperature, pressure, and water consumption. High resolution (0.2 nm) diffraction patterns were recorded from unfixed, unstained, fully hydrated catalase crystals. This is a good functional test of DIFPEC operation and specimen preparation because catalase disorders irreversibly if exposed to less than 95% relative humidity. High- and low-angle diffraction patterns were recorded from unfixed, unstained, fully hydrated rat hemoglobin crystals. All patterns were recorded using 10−4 C/cm2. Whole cell mounts prepared in different ways were imaged and show that 5.3 kPa of nitrogen gas has no detrimental effect on image contrast or resolution. The column vacuum and differentially pumped volume pressure were measured for a number of pumping configurations, as well as for several gases and pressures in the specimen volume. The pressure in the high vacuum portion of the objective lens gap is conductance-limited when the DIFPEC is in place.

Dynamical Scattering in Electron Diffraction of wet Protein Crystals

The significance of dynamical scattering effects remains the major question in the structural analysis by electron diffraction of protein crystals preserved in the hydrated state. In the few cases (single layers of purple membrane and 400-600 Å thick catalase crystals examined at 100 kV acceleration voltage) where electron-diffraction patterns were used quantitatively, dynamical scattering effects were considered unimportant on the basis of a comparison with x-ray intensities. The kinematical treatment is usually justified by the thinness of the crystal. A theoretical investigation by Ho et al. using Cowley-Moodie multislice formulation of dynamical scattering theory and cytochrome b5as the test object2 suggests that kinematical analysis of electron diffraction data with 100-keV electrons would not likely be valid for specimen thickness of 300 Å or more. We have chosen to work with electron diffraction patterns obtained from actual wet protein crystals (rat hemoglobin crystals of thickness range 1000 to 2500 Å) at 200 and 1000 kV and to analyze these for dynamical effects.

A simple method of optical filtration

A simple method is described for analyzing an object (or its image) in order both to reveal and filter out an intrinsic periodicity in the object itself. The method significantly improves the imaging of fine details in periodic structures. Effects of “multiplication”, caused by diffraction during observation through a fine-mesh grating, and of subsequent superposition of the diffracted rays onto neighboring details of the intrinsic periodicity have been used. The method is illustrated by results obtained with untreated and filtered electron micrographs of bovine liver catalase crystal, and of the Z -line region of the myofibril of cross-striated fish muscle.

Proteinaceous crystals in cells of virus infected plants

Crystal-containing organelles in cells of virus infected plants lying at chloroplasts and mitochondria are identical with single membrane-bound microbodies containing crystals of catalase described in healthy plants. Massive complex inclusions caused by turnip mosaic virus very frequently contain the same microbodies with crystal inclusions; that phenomenon may be related to some pathophysiological changes of virus infected plants. Comparable proteinaceous crystals, but not lying within microbodies limited by a membrane, may also be found in cytoplasm of infected cells. These crystals are sometimes surrounded by a substance resembling the microbody matrix. Disintegrated cytoplasm of virus infected cells may also contain the same crystals lying free in “empty spaces”. Cytopathological effects responsible for this phenomenon and possible artifacts as well are discussed.

Crystallographic evidence for the structural isomorphism of deer and beef catalase

2 crystal forms of deer liver catalase have been grown and analyzed by X-ray diffraction. Both are of space group P212121 with cell dimensions I: a=88.5 A, b=140.5A and c=235 A; II: a=124 A, b=128 A and c=154 A. The first of these 2 forms appears to be virtually isomorphous with crystals of beef liver catalase.

Electron microscopy of frozen hydrated biological specimens

The use of frozen hydrated specimens for molecular structure determination is limited primarily by radiation damage. The radiation damage effect in frozen hydrated catalase crystals has been measured in terms of the loss of electron diffraction. The results show an improvement for this type of specimen relative to wet hydrated or to glucose embedded catalase crystals at room temperature. Bright field images of unstained, frozen hydrated catalase crystals extend to a resolution of 11.5 A. The image resolution is presently limited by design problems with the liquid nitrogen cooled specimen stage. Rather high contrast is present in these images of unstained biological material, presumably due to the 30% difference in mass density between ice and protein. The improved resistance to radiation damage also makes it possible to give better statistical definition to the image, thereby making the image features easier to see.

The aerodynamic diameter of branched chain-like aggregates

The aerodynamic behaviour of branched chain-like aggregates of iron oxide as well as gold particles is studied using a centrifugal aerosol spectrometer according to Stöber and Flachsbart. The aerosols, which were radioactively labeled, were produced by an exploding wire. The size distribution of the primary particles of the aggregates is determined by means of electron microscopy with negatively stained catalase crystals as an internal length standard. A relation between the aerodynamic diameter ( d a ) and the number of primary particles ( n) of the aggregates is obtained. This relation can be divided in a region for which d a is proportional to n 1/6 and a region for which d a is proportional to n 1/3. There is a significant accordance between these results and the results obtained by Stöber for aggregates of monodisperse polystyrene spheres.

Radiation damage in stained catalase at low temperature.

Radiation damage, which occurs in the beam of the electron microscope, has been studied in uranyl acetate stained crystals. Damage is observed in terms of the disappearance of higher orders of the electron diffraction pattern. In this study it has been found that the damage at very low specimen temperatures proceeds both more rapidly and to a greater final degree than is the case at room temperature.

Thickness measurements of wet protein crystals in the electron microscope

With the stable environment afforded by a dynamically pumped wet specimen chamber, the thickness of a fully hydrated protein microcrystal may be obtained in an electron microscope from an incident-beam attenuation measurement after disappearance of the diffraction pattern. Such measurements on orthorhombic catalase crystals have been successfully cross-correlated with thickness measurements on the same crystals made with an interference light microscope over a range from a few hundred to a few thousand Ångströms.

Electron Microscopy of Frozen, Hydrated Biological Specimens

Recently, a method has been developed in this laboratory for maintaining the hydration of biological materials by using frozen specimens. With this technique specimens are prepared using folding grids on which are mounted composite support films of carbon and SiO. The technique essentially embeds the biological material in a thin aqueous film between the two supporting films. The sandwiched specimens are then frozen directly in liquid nitrogen. While it was thought at first that freezing directly in liquid nitrogen might cause disordering of periodic or crystalline biological specimens, this has been shown not to be the case. High resolution electron diffraction patterns have been obtained from frozen unstained catalase crystals.The microscopy of frozen specimens depends on their resistance to damage by electron irradiation. The critical exposure for fading of the electron diffraction intensities from frozen unstained catalase crystals has been measured.