Micro-determination of amino acid composition of proteins electroblotted onto polyvinylidene difluoride membranes

A method for determination of amino acid composition of proteins separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes is described. A single blotted band containing 50 to 200 pmoles of protein was cut out and submitted to acid hydrolysis with HCl followed by derivatization with phenylisothiocyanate. The amino acid derivatives were separated by reverse phase high-performance liquid chromatography. Bovine serum albumin, lysozyme, myoglobin, ovalbumin, soybean trypsin inhibitor and carbonic anhydrase were analyzed; the results revealed a good correspondence with reported values. This can be considered an analytical method to determine the amino acid composition of samples from microquantities of protein mixtures, particularly in those cases in which SDS-polyacrylamide gel electrophoresis is the most suitable separation system.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.     

Nature of the tryptic/chymotryptic inhibitor from horsegram (Dolichos biflorus).

A protease inhibitor specific to trypsin and chymotrypsin was purified from horsegram (Dolichos biflorus) with the inhibition index 0.24 micrograms/micrograms for trypsin and 0.36 micrograms/micrograms for chymotrypsin. In SDS-PAGE, the inhibitor protein was seen as a single band with apparent molecular mass Mr = 15,500. However, on fast protein liquid chromatography (FPLC) or non-denaturating PAGE, the inhibitor resolved into four components revealing the existence of isoinhibitors. Data on amino acid analysis indicate that the isoinhibitors are closely related. The major amino acids in the inhibitor are half cystine (18.9 mole %), aspartic acid (12.7 mole %) and serine (14.3 mole %). The inhibitor was partially stable to 0.1% sodium dodecyl sulphate, 8M urea or 6M guanidine hydrochloride. The inhibitory activity was lost on reduction or carboxamidomethylation or acetylation. Modification of the arginine groups or CNBr cleavage of the protein did not result in significant loss of either tryptic or chymotryptic inhibitory activities. The isoinhibitors separated by FPLC reacted with polyclonal antibody raised in rabbits and had pI values ranging from 4.8-5.1. The horsegram inhibitor thus resembles other Bowman-Birk protease inhibitors.     

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Amino acid analysis on polyvinylidene difluoride membranes

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.     

Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes.

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.     

Identification of a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of lizard fish Saurida wanieso which specifically cleaves the arginine site

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

The direct hydrolysis of proteins containing tryptophan on polyvinylidene difluoride membranes by mercaptoethanesulfonic acid in the vapor phase.

A procedure for the amino acid analysis of polypeptides that contain tryptophan on polyvinylidene difluoride membranes is described. Lysozyme, carbonic anhydrase, phytochrome, and ovalbumin were tested. The protein, which was separated from others by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was blotted from the gel onto a polyvinylidene difluoride membrane and directly hydrolyzed by 3 N mercaptoethanesulfonic acid vapor in a vacuum at 176 degrees C for 25 min. The hydrolysate was extracted with 0.1 N HCl and 30% methanol and used for amino acid analysis. The tested proteins were adequately hydrolyzed, and the recovery of tryptophan was very efficient.     

Purification and amino acid analysis of plasma acid stable trypsin inhibitor

Acid stable trypsin inhibitor (ASTI), with a molecular weight of about 85,000 by gel filtration, specific activity of 1,498 U/mg protein and pI of 1.6, from renal failure patient plasma was first purified. The amino acid composition of the purified ASTI was found to be that of a Gly- and Glu-rich protein which lacked His, closely resembling that of urinary trypsin inhibitor. The NH2-terminal amino acid sequence was Ala-Val-Leu-Pro-Gln-Glu- Glu-Glu-Gly-X-Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-Lys-Lys-Glu- Asp-Ser-Ser-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.     

Quantitation of the major proteins of the human erythrocyte membrane by amino acid analysis

The proteins of the human erythrocyte membrane have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resulting gel cut into 2-mm sections, and the amino acid content and composition of each slice measured using a sensitive method of amino acid analysis. The distribution of proteins among bands coincides closely with that estimated using staining intensity. Composition data for the major bands agree well with those reported for the purified proteins in all cases except that of band 4.5. Using quantitative amino acid analysis and resistive particle counting the total protein content of purified membranes was found to be 3.75 X 10(-13) g/cell, which is substantially less than previous estimates based on indirect methods. These data are used to calculate the number of copies of each major protein in a single erythrocyte.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.     

The role of chloroplast-membrane-protein synthesis in the circadian clock. Purification and partial characterization of a polypeptide which is suggested to be involved in the clock.

A polypeptide (polypeptide P39), which is presumed to involved in the photosynthetic circadian rhythm in the green alga Acetabularia, was purified from the EDTA-insoluble chloroplast membrane fraction by means of preparative dodecylsulfate gel electrophoresis and then partially characterized. The purity of the isolated polypeptide P39 was confirmed by a further electrophoresis on an analytical dodecylsulfate gel and further elucidated by amino-terminal analysis which shows that glycine is the only amino-terminal amino acid of the purified polypeptide material. The molecular weight of the polypeptide P39 was found to be about 39,000 on analytical gel electrophoresis and the value was further supported by those obtained from amino acid composition and peptide mapping. The amino acid composition of polypeptide P39 showed that the proportion of intermediate amino acid groups is high while the proportion of hydrophilic amino acid groups is well balanced by that of hydrophobic amino acid groups, a property characteristic of membrane proteins.     

Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.