Formation of a steady state in the radiolysis of ferrimyoglobin in aqueous solution

The interaction of radiation-generated · OH radicals with ferrimyoglobin in deaerated aqueous solution at neutral pH has been quantitatively studied. Changes in the visible absorption spectrum have been analyzed on the basis of composition changes of the ferri, deoxy, and ferriperoxide forms of the metalloprotein. A postirradiation thermal process must be considered in order to evaluate the radical-induced composition changes. Initially, ·OH induces reduction of ferrimyoglobin to the deoxy form with a G value (molecular yield/100 eV of absorbed energy) in the zero-dose limit of 1.4 (±0.2). Radiation-generated H₂O₂ reacts with the ferrimyoglobin substrate to produce ferrimyoglobin peroxide with a G value of 0.7 (±0.1) in the zero-dose limit. At doses of >1 krad μm^?1 of myoglobin present, the composition of the three myoglobin derivatives reaches a radiolysis steady state. In this moderate-dose plateau region, this composition is 44% ferri, 18% deoxy, and 38% ferri peroxide. The · OH-induced hemoprotein radicals that do not initiate 1-eq redox conversions undergo reactions that generate dimer and other globin-modified material.     

Absorption spectra of sperm-whale ferrimyoglobin

1. Sperm-whale ferrimyoglobin was found to contain 0.308% of Fe, on a dry weight basis, corresponding to a molecular weight of 18130. The solid takes up moisture to an equilibrium state, and was then assayed to contain 0.280% of Fe. 2. Absorption spectra are presented for acidic ferrimyoglobin, Fe(+)(H(2)O), and its conjugate base, Fe-OH, as well as for the fluoride and cyanide complexes, within the range 200-2500mmu. Data for ferromyoglobin-carbon monoxide, Fe(II)-CO, in the visible region are also included. 3. Minor spectral differences have been found between whale and horse myoglobins, particularly in the effect of temperature on the visible-absorption spectrum of Fe-OH.     

Crystallization and Physical Characterization of Porcine Myoglobin

Porcine myoglobin was isolated by fractionation with ammonium sulfate and crystallized with 3.8 M phosphate buffer.

Chromatography of the porcine crystalline myoglobin on a molecular-sieve column gave a single elution band, which agreed well with that of horse myoglobin.

By ultracentrifugal analysis, the sedimentation constant (s_20,w), was found to be 1.96 S.

The iron content of the myoglobin was 0.324%. This value corresponds to the minimal molecular weight of 17,200.

Absorption curves and millimolar extinction coefficients of the porcine myoglobin in several derivatives, namely, reduced (RMb), met (MMb), carboxy (MbCO) and cyanment (MMbCN) forms were examined in the visible region. The spectral characteristics of the porcine myoglobin derivatives were in good agreement with those of horse myoglobin,

Three components were detected for the porcine myoglobin by starch gel electrophoresis, and each of them had higher migration velocity than those of horse myoglobin.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L). Identification of the trypsin-reactive site, partial-amino-acid sequence and further physico-chemical properties of the major inhibitor.

The two principal isoinhibitors P-5 and P-6 isolated earlier from the seeds of chick peas (Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix-bound trypsin are shown to be the virgin and trypsin-modified forms of the same inhibitor. The virgin inhibitor P-5 consists of a single peptide chain of 66 amino acid residues cross-linked by seven disulfide bridges. Upon interaction with the matrix-bound trypsin under the conditions used the virgin inhibitor P-5 is about 50% converted to P-6 by cleavage of a single Lys-Ser linkage at position 14-15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1-14 and 15-66 separate readily by molecular-sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence 1-25 was established by Edman degradation.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Studies on myoglobin from the finback whale (Balaenoptera physalus). Preparation, physicochemical and immunochemical characterization, differentiation from sperm-whale myoglobin, amino acid composition and end-terminal analyses

1. Crystalline myoglobin was isolated from the skeletal muscle of the finback whale and fractionated, in its cyanmet form, into nine components (I-IX) by chromatography on CM-cellulose. Also in the cyanmet form, it was resolved into six components by electrophoresis on starch gel. Correspondence between the electrophoretic and chromatographic components was determined, and interconversion between components revealed by chromatography and electrophoresis. 2. The chromatographic myoglobin components were homogeneous in the ultra-centrifuge. Molecular weights of certain components were determined by means of sedimentation equilibrium and by gel filtration on Sephadex G-100. Values from these two methods corresponded to the minimum molecular weight calculated from the iron content. 3. The spectral properties of the chromatographic components were investigated in the visible and the ultraviolet ranges. 4. The major components of finback-whale myoglobin and sperm-whale myoglobin showed almost identical spectral, electrophoretic and chromatographic behaviours, but had different infrared spectra. The infrared spectra of the corresponding apoproteins were almost identical. 5. Rabbit antisera to sperm-whale myoglobin component X cross-reacted with finback-whale myoglobin components V, VI and VII only about 30%. 6. The major chromatographic components of finback-whale myoglobin have identical amino acid compositions. The polypeptide chain contains 151 amino acid residues and its molecular weight is 17504. 7. The N-terminal end of the chain is: [Formula: see text] Amino acids released from myoglobin by the action of carboxypeptidase A at different intervals were determined.     

Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.     

Evidence that chromium is an essential factor for biological activity of low-molecular-weight, chromium-binding substance

Biologically active Low-molecular-weight, chromium-binding substance (LMCr) has been isolated from the liver of rabbits injected with potassium bicarbonate and cow's milk. This substance enhances glucose oxidation and lipogenesis from glucose in rat adipocytes [Yamamoto A. et al. (1987) Eur. J. Biochem. 165, 627-631; (1988) J. Nut. 118, 39-45]. LMCr was shown to lose its activity almost completely as an effectant of glucose metabolism when Cr was deleted under acidic conditions and separated from LMCr by EDTA-chelation and successive molecular-sieve chromatography. Reincorporation of Cr3+ into apo-LMCr resulted in 50-90% recovery of its original activity. These findings suggest that the biological activity of LMCr resides in Cr.     

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.     

Isolation and some structural analyses of a proteodermatan sulphate from calf skin.

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).     

Improved method for purification of human Escherichia coli heat-stable enterotoxin by hydrophobic interaction, molecular-sieve and high performance ion exchange chromatography

A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 degrees C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity.     

Analytical exclusion chromatography

This review summarizes the development of exclusion chromatography, also termed gel filtration, molecular-sieve chromatography and gel permeation chromatography, for the quantitative characterization of solutes and solute interactions. As well as affording a means of determining molecular mass and molecular mass distribution, the technique offers a convenient way of characterizing solute self-association and solute-ligand interactions in terms of reaction stoichiometry and equilibrium constant. The availability of molecular-sieve media with different selective porosities ensures that very little restriction is imposed on the size of solute amenable to study. Furthermore, access to a diverse array of assay procedures for monitoring the column eluate endows analytical exclusion chromatography with far greater flexibility than other techniques from the viewpoint of solute concentration range that can be examined. In addition to its widely recognized prowess as a means of solute separation and purification, exclusion chromatography thus also possesses considerable potential for investigating the functional roles of the purified solutes.     

Characterization of a camel milk protein rich in proline identifies a new beta-casein fragment

A camel milk whey protein has been isolated by reverse-phase high performance liquid chromatography. The protein is, like caseins, rich in proline (25% of the whole protein). The N-terminal amino acid sequence shows that the protein is homologous with a C-terminal region of beta-caseins analyzed from other species. The protein is concluded to be a fragment of beta-casein, derived from a non-tryptic type of cleavage of the parent molecule, and increasing the multiplicity of known casein products.     

Estimation of protein molecular weights by high performance molecular-sieve chromatography on agarose columns in sodium dodecyl sulphate solution

Calibration curves showing the linear relationship between -log KD and (molecular weight)2/3 are presented for proteins subjected to molecular-sieve chromatography in a 0.1% sodium dodecyl sulphate solution on crosslinked gels of different agarose concentrations (5, 9, 12 and 20%). These gels permit separation and estimation of molecular weights of proteins in the range 5000 to about 400 000, 130 000, 130 000, and 100 000, respectively. Separations of model proteins, heavy and light chains of gamma-globulin, and cyanogen bromide fragments of cellulase are shown.     

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.     

Proteomic identification of camel seminal plasma: Purification of β-nerve growth factor

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.