An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

Free radical induced inactivation of creatine kinase: sites of interaction, protection, and recovery

The study aims at a clarification of the oxidative damage of creatine kinase isoenzymes by X-ray-induced water radiolysis. The radical species generated by this method (under appropriate conditions) are similar to those discussed in the context of mitochondrial energy metabolism. The decay of the enzyme activity is accompanied by a strong decrease of the number of accessible SH groups and by a reduction of the endogenous tryptophan fluorescence. Free radical effects are diminished if irradiation is carried out in the presence of 2-mercaptoethanol. Partial recovery of the activity (repair) is observed if 2-mercaptoethanol is added after irradiation. The experiments suggest a twofold importance of thiol reagents (RSH): to reduce the concentration of free radicals by scavenger reactions and to modify the inactivation mechanism in such a way that efficient repair of enzyme damage may be achieved. Cysteine 282 of MM-CK (Cys-278 in the case of Mi-CK) seems to play a crucial role in this respect. Blockage of the SH group of cysteine 282 by oxidized glutathione effectively protects the enzyme against inactivation by NO(*)(2) radicals. In the absence of nitrogen dioxide and of thiol reagents, however, inactivation seems to proceed via a less specific mechanism involving additional targets of the enzyme.     

Partial puridication of a membrane protein from human erythrocytes involved in glucose transport.

In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Combinatorial chemical reengineering of the alpha class glutathione transferases

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.     

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime — bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound protein was recovered by incubating the adsorbent with 2-mercaptoethanol. Crude antibody and uterine cytosol was prepared for affinity chromatography in buffer containing 10^?3 to 10^?2M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S ag SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200–300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.     

Topography of the C. coli 5S RNA-protein complex as determined by crosslinking with dimethyl suberimidate and dimethyl-3,3''-dithiobispropionimidate

5S RNA-protein complexes were prepared in vitro using partially purified E. coli 5S RNA and total E. coli 70S ribosomal proteins. The complexes were isolated from sucrose gradients and shown to contain proteins L5, L18, L25 and a fourth protein not heretofore characterized and designed L31. The complexes were treated with the crosslinking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate. Both reagents gave identical patterns of crosslinked proteins when analyzed by one-dimensional polyacrylamide/dodecylsulfate gel electrophoresis. Dimers of L5-L31', L5-L18 and L18-L18 and a trimer containing L5, L18 and L31' were identified by diagonal polyacrylamide/dodecylsulfate gel electrophoresis of the proteins crosslinked with dimethyl-3,3'-dithiobispropionimidate. No crosslinking was detected between L25 and the other three proteins.     

Changes in antigenic properties of the p35 protein of vaccinia virus in various protein fractions of the virion with the use of monospecific antisera

Three monospecific antisera to the major 35 kD (p35) surface protein of vaccinia and ectromelia viruses have been obtained. Two of them are obtained to p35 protein isolated by electrophoresis in the presence of sodium dodecylsulfate from the protein fractions of vaccinia virus, soluble in NP40 and NP40 with dithiothreitol (NP40 and DTT-fractions). The third serum is obtained to NP40-fraction of ectromelia virus, containing practically only p35 protein. The obtained antisera were compared in the reactions with the different fractions of viral proteins in two versions of solid phase radioimmunoassay. The effect of such reagents as sodium dodecylsulfate, NP40, 2-mercaptoethanol, ethanol on the antigenic properties of p35 protein from vaccinia virus is discussed.     

Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins

The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be carried out in a typical chemistry laboratory, usually taking about 2-3 d. A wide range of other biarsenical reagents and intermediates that also bind to tetracysteine-tagged (CysCysProGlyCysCys) proteins can be prepared similarly using this general procedure.     

Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.     

Preparation of 1-thioglycosides having ω-aldehydo aglycons useful for attachment to proteins by reductive amination

A series of 1-thioglycosides containing an ω-aldehydo group (as the dimethyl acetal) on the aglycon were prepared by reaction of O-acetyl-1-thioaldoses with N-(chloroacetyl)aminoacetaldehyde dimethyl acetal, a compound readily prepared by the action of chloroacetyl chloride or chloroacetic anhydride on 2-aminoacetaldehyde dimethyl acetal. O-Deacetylation of the 1-thioglycosides, followed by deacetalation, yielded the desired products. An analogous 1-thioglycoside having a longer aglycon was prepared by reaction of 1-thio-D-galactose with (6-aminohexanoyl)-aminoacetaldehyde dimethyl acetal, obtained by condensing 6-bromohexanoic acid and aminoacetaldehyde with 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide. These glycosides were found to be useful for modification of proteins to yield neoglyco-proteins.     

Uptake of alpha-ketoisocaproic acid in lymphoblast line WI-L2.

The uptake of α-ketoisocaproate by the cultured human lymphoblast line WI-L2 appears to be mediated by a transport system which has an apparent Km of 125 μM. The rate of uptake of α-ketoisocaproate decreases with increasing pH values, i.e., pH 6 > 7 > 8 and is stimulated by sodium at all pH values. Closely related branched chain α-ketoacids, α-keto-β-methylvaleric and α-ketoisovaleric exhibited the greatest inhibition of α-ketoisocaproate transport. Straight chain α-keto acids inhibited α-ketoisocaproic acid uptake to a lesser degree as did the α-hydroxy analogs of the branched chain α-keto acids. Inhibitors of the general anion transport system of erythrocytes, 1-anilino-8-napthalene sulfonic acid and 4-acetamido-4-isothiocyanostilbene-2-1′-disulfonic acid did not affect α-ketoisocaproate transport. A reduced sulfhydryl group is critical for α-ketoisocaproate acid uptake; transport is partially or completely inhibited by sulfhydryl reagents such as dithio-bis-nitrobenzoate, iodoacetamide, and p-chloromercuribenzoate. Inhibition by the sulfhydryl reagents is reversed with β-mercaptoethanol or partially with dithiothreitol.     

Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents

We encountered β-mercaptoethanol-dependent artifact signals in western blot analyses using polyclonal antisera. Replacing β-mercaptoethanol with dithiothreitol in the loading buffer did not eliminate the artifact signals. However, lowering the concentration of either dithiothreitol or β-mercaptoethanol eliminated the background problems and allowed specific detection of the target protein. These results are consistent with the background signal being caused by anti-keratin antibodies in the antisera and keratin contamination of reagents. This study highlights the importance of testing a range of reducing agent concentrations when trying to eliminate artifact bands from western blots. However, this method may not be applicable when target proteins have disulfide bridges.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Organization of synaptic junction proteins

Most of the polypeptides of isolated brain synaptic junction preparations are cross-linked by disulfide bonds; these bonds are readily reformed following reduction by β-mercaptoethanol, suggesting that other, non-covalent interactions may hold the polypeptides in close contact. When synaptic junctions were treated with β-mercaptoethanol in conjunction with a large variety of reagents known to disrupt certain types of non-covalent bonds, however, the polypeptides could still be cross-linked. Furthermore, virtually all of these species remained associated with β-mercaptoethanol-reduced membrane in the presence of 0.05 N NaOH or 0.5% Triton X-100, although each of these solvents extracts large amounts of protein from extra-junctional membrane, which has a composition apparently similar to that of synaptic junctions.These results indicate that any non-covalent interactions existing among synaptic junction proteins are inaccessible to treatments at the surface of the membrane, being located within the lipid bilayer or perhaps at points of contact with sub-membranous arrays such as the post-synaptic density. Under special conditions, the polypeptides of extra-junctional membrane can also be induced to form disulfide cross-links among one another, and these bonds can likewise be rapidly reformed following reduction, giving this membrane properties similar to that of the junction. This suggests a model by which stabilized junctional membrane may be formed from fluid extra-junctional membrane during synaptogenesis.     

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.     

Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma)

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding β-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of β-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.     

Identification of retrovirus matrix proteins by lipid-protein cross-linking.

Dimethyl suberimidate and its analogs are symmetrical bifunctional reagents that form amidine linkages with primary amino groups. These reagents have been used previously to study nearest neighbor relationships of proteins in viruses and in complex structures such as ribosomes. Dimethyl suberimidate also reacts with phosphatidylethanolamine, which can be radioactively labeled specifically with [¹⁴C]ethanolamine. When enveloped viruses containing radioactive phosphatidylethanolamine are exposed to the diimido ester, a fraction of the radioactivity becomes linked to viral structural proteins. Upon separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the lipid-protein complexes can be visualized on fluorograms of the gels. The cross-linking of lipids to proteins is specific, since it requires the viral structure to be intact, and since only certain proteins become chemically linked to phosphatidylethanolamine even though all the proteins react with dimethyl suberimidate. In vesicular stomatitis virus, the structure of which has been well characterized, only the glycoprotein and the matrix protein become linked to lipid. This is consistent with their known locations protruding outwards and inwards from the virus membrane, respectively. Thus we infer that the cross-linking technique can be used to identify proteins in close proximity to the lipid bilayer. In the avian leukemia and sarcoma viruses the protein designated p 19, and in the murine leukemia viruses p 15 become linked to radioactive lipid. Since avian p19 and murine p15 are internal structural proteins, we infer that they are equivalent to the matrix protein defined for other other enveloped viruses.     

Reconstitution of the diiron sites in hemerythrin and myohemerythrin

The first reconstitutions of functional diiron sites in the nonheme O2-carrying proteins hemerythrin (Hr) and myohemerythrin (myoHr) have been achieved. Both proteins are reconstituted under anaerobic conditions, and the procedure consists of (i) denaturation of the native met form with 6 M guanidinium chloride in the presence of sodium dithionite and 2,2'-dipyridyl, (ii) separation of the apoprotein from the other reagents and products, (iii) addition of an iron(II) stock solution to the apoprotein in the presence of 2-mercaptoethanol, and (iv) several cycles of slow dilution and reconcentration by ultrafiltration to remove excess reagents. Iron analyses indicate that the apoproteins have been essentially completely freed of iron and that reconstituted Hr contains its full complement of iron, i.e., approximately 2 Fe/subunit. Ferrous rather than ferric iron appears to be necessary for recovery of the native structures for both myoHr and Hr. In the case of Hr, reconstitution was successful only when iron(II) was added to apoHr prior to removal of denaturant. ApoHr is essentially insoluble at pH 7 in the absence of denaturants but remains soluble when denaturant is removed in the presence of ferrous iron, which leads to recovery of the octameric structure containing all of its diiron sites. Iron(II) apparently stabilizes the native or a nearly native structure during reconstitution. OxymyoHr and oxyHr are the major initial products of reconstitution. The yield of oxymyoHr from apomyoHr was approximately 87%. In contrast to reconstituted oxymyoHr, where essentially all of the iron appears to be functional, approximately 30% of the diiron sites in the reconstituted oxyHr are unable to bind O2 at ambient p(O2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Deposition of Matrix and Crystalloid Storage Proteins during Protein Body Development in the Endosperm of Ricinus communis L. cv. Hale Seeds

Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of which the crystalloid proteins make up approximately 70%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the crystalloids are a family of at least four proteins which reduce to two complementary groups after 2-mercaptoethanol treatment. The matrix, which comprises the remainder, has two major components, the soluble albumins and the lectins. The lectins are the only glycoproteins within the mature protein body. Both cytochemical staining and SDS-PAGE indicate that the synthesis of the crystalloid and the majority of matrix proteins begins some 20 days after pollination. Additionally, the crystalloid proteins are synthesized concurrently, whereas there is temporal variation in the synthesis of matrix proteins.