Periodate oxidation of methionine in proteins

Treatment of free methionine with an equimolar amount of periodate gave nearly quantitative formation of the sulfoxide; treatment of free methionine sulfoxide with equimolar periodate gave nearly equal amounts of the original sulfoxide and the sulfone. Treatments of 0.5-1.0% solutions of the following proteins with relatively low concentration of periodate (5 mm) gave the following approximate values for conversion of methionine sulfoxide from total methionine: bovine pancreatic ribonuclease A (2 of 4), chicken ovalbumin (14 or 17), chicken ovotransferrin (5 of 11), human serum transferrin (2 of 8), bovine α-chymotrypsin (1 of 2). It is recommended that when proteins are treated with sodium periodate (and probably with oxidizing agents in general), especially when changes in properties are observed, determinations of methionine sulfoxide should be done.     

The purification of peptides which contain methionine residues

A novel procedure for isolating peptides which contain methionine is described. It relies upon the reversible increase in charge which occurs upon the alkylation of methionine by iodoacetamide. A digest of the protein is reacted with lodo[¹⁴C]acetamide under conditions which direct the reaction exclusively to the methionine residues. In this way, methionine-containing peptides are rendered radioactive and gain one positive charge per methionine simultaneously. The digest is then separated on a cation exchange column, the peptides are located by their radioactivity, and they are separately collected. The carboxyamidomethylation is reversed by thiolysis, which eliminates the extra positive charge which each methionine-containing peptide bore, decreasing their charge selectively. A second chromatographic separation, performed on the same cation exchange column, is sufficient to produce the desired peptides in a high state of purity. Equine myoglobin and bovine ribonuclease were used as models to demonstrate the feasibility of this approach. Methionine-containing tryptic peptides were purified from digests of these proteins in yields which were equivalent to those of previously reported separations. The present procedure, however, is applicable to peptide mixtures of far greater complexity than those which were derived from the model compounds and can be applied with the same success to digests of very large proteins containing many methionine residues.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

Proteolysis of human reticulocyte membrane proteins: Evidence for a physiological role of the acid endopeptidases

Endogenous membrane proteins, labeled by incubating human reticulocytes with l-[¹⁴C]leucine, are degraded at pH 7.3 by membrane-bound acid proteinases. Solubilized membrane proteins are also degraded at neutral pH by the purified membrane acid proteinases. Exogenous proteins are not degraded by intact membranes and therefore association with the membrane seems to be an essential requirement for protein degradation in the physiological pH range. These findings provide evidence for a physiological function of the enzymes previously characterized as acid proteinases.     

Modification of available arginine residues in proteins by p-hydroxyphenylglyoxal

p-Hydroxyphenylglyoxal reacts with arginine residues in proteins to give a single product which can be quantitated spectrophotometrically. The reaction takes place under mild conditions, pH 7–9 and 25°C. Under these conditions up to complete modification of N^α-citraconyl-l-arginine was obtained within 60 min with less than 5% modification of other common amino acid side chains. The extent of modification in a protein can be determined at 340 nm using the molar absorption coefficient of 1.83 × 10⁴m^?1 cm^?1 for the product at pH 9.0 and 25°C following removal of excess reagent by gel filtration. Several proteins, previously shown to have essential arginines, were modified by p-hydroxyphenylglyoxal and the losses in arginines were determined spectrophotometrically. These results were in close agreement with those of previous investigators. Rhea ovomucoid, a glycoprotein without arginines but containing an essential lysine, was relatively unaffected.     

Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal

A new method is presented for the colorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 m sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer's law in the range 0.03–0.33 mm arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.     

Second-derivative spectroscopy of proteins: Studies on tyrosyl residues

Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.     

A critical physiological role of zinc in the structure and function of biomembranes

Of the large number of recognized zinc metalloenzymes, the activities of only a few are significantly decreased in severely zinc deficient animals. On the other hand, physiological pathology is manifested rapidly after dietary zinc deprivation. This shows that zinc exerts physiological and biochemical roles other than as a component of the known zinc metalloenzymes. The research results reviewed here suggest that zinc plays an important role in the maintenance of membrane structure and function.     

A specific micromethod for determination of acetyl residues in proteins

A method is described for the separation and concentration of rat liver lysosomes from mitochondria in a one-step procedure by zonal centrifugation. Some of the practical problems associated with the use of the B-XX1X rotor are discussed.     

Collagen-induced platelet aggregation and release. I effects of side-chain modifications and role of arginyl residues

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol.

A solvolytic N-desulfation of heparin was developed by treatment of its pyridinium salt with dimethyl sulfoxide containing 5% of water or methanol for 1.5 h at 50 degrees. Chemical and chromatographic studies showed that the solvolytic desulfation is a useful method for N-desulfation of heparin without depolymerization of the heparin molecule. The partially N-desulfated heparins were also obtained by treatment with dimethyl sulfoxide containing 5% of water at 20 degrees, and their anticoagulant activity is related to the degree of N-desulfation.     

Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol.

A solvolytic desulfation of glycosaminoglycuronan sulfates was developed by treatment of their pyridinium salts with dimethyl sulfoxide containing 10% of water or methanol at 80-100 degrees. Chemical and physical studies showed that the solvolytic desulfation is a useful method applicable to all the known glycosaminoglycuronan sulfates without producing depolymerization or unfavorable chemical changes in the polysaccharide molecules. An almost completely desulfated, N-acetylated heparin (S: 0.12%) was obtained by treatment of an N-desulfated and N-acetylated heparin with dimethyl sulfoxide containing 10% of methanol for 2 h at 100 degrees.     

Cryopreservation of Giardia lamblia with dimethyl sulfoxide using a Dewar flask

This study examined the effect of varying freezing conditions on the human intestinal parasite Giardia lamblia (Portland-1 strain) using a constant vacuum in a Dewar flask and an ethanol bath to regulate the cooling rate. The cryopreservation of the trophozoite stage was investigated. Dimethyl sulfoxide (Me2SO), the cryoprotective agent of choice, was added directly to Giardia growth medium. Me2SO toxicity assays were conducted on those concentrations used in the freezing protocol. The results of this study indicated a 6.5% (v/v) Me2SO concentration yields a 90% survival based upon organism motility. A 30.9% cell viability was obtained by freezing in medium without a cryoprotective agent. Recommendations are offered concerning alternate viability criteria.     

Preparation of methyl glycosides of di- and higher oligosaccharides from glycosaminoglycuronans by solvolysis with dimethyl sulfoxide containing methanol

Solvolysis of chondroitin 4- or 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of methanol for 18 h at 95° resulted in the cleavage of the 2-amino-2-deoxy-D-glucoside bonds together with initial desulfation to give methyl β-glycosides of N-acetylchondrosine as a main product and, in addition, higher oligosaccharides, without any loss of uronic acid. Dermatan sulfate was also depolymerized to yield methyl glycosides of di- and higher oligosaccharides under the same conditions. Hyaluronic acid (free acid) was depolymerized by the same solvent in the presence of an equimolar amount of pyridine-sulfur trioxide or pyridinium sulfate per disaccharide unit to give methyl glycosides of di- and higher oligosaccharides. In contrast N-desulfated, N-acetylated heparin was stable under these solvolytic conditions and did not yield heparin oligosaccharides.     

Calorimetric studies of flavin-binding proteins: FMN and FAD binding to hen egg riboflavin-binding proteins

Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 10⁵ and 10⁴ were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form.     

Cell surface proteins of Drosophila. II. A comparison of embryonic and ecdysone-induced proteins

The cell-surface proteins of Drosophila embryos at gastrula and myoblast fusion stages were characterized by radioiodination and two-dimensional gel electrophoresis. Over 13% of the cell surface proteins detected in gastrula embryos were not found in myoblast fusion stage embryos or in Drosophila embryonic cell line EH34A3 cells. Nearly 18% of the cell-surface proteins detected in myoblast fusion stage embryos were evident only at that stage. Embryonic cell-surface proteins were compared with cell-surface proteins from untreated EH34A3 cells and EH34A3 cells treated with 20-hydroxyecdysone, which induces cell aggregation and the expression of "new" proteins at the cell surface (D. F. Woods and C. A. Poodry, 1983, Dev. Biol. 96, 23-31). Only one of the proteins induced by ecdysone in EH34A3 cells was detected in the NP-40 soluble fraction of radioiodinated cell lysates, even after fractionation by lectin affinity chromatography and immunoprecipitation to enrich for putative ecdysone induced proteins. However, extraction of the NP-40 insoluble pellet of embryo cells revealed one additional protein that was present both in myoblast fusion stage embryos and hormone-treated culture cells. It was concluded that except for these two proteins, the cell-surface proteins induced in cultured cell lines by treatment with 20-hydroxyecdysone are not present in significant amounts in gastrula or myoblast fusion stage embryos.