A novel method for selective isolation of C-terminal peptides from tryptic digests of proteins by immobilized anhydrotrypsin: application to structural analyses of the tail sheath and tube proteins from bacteriophage T4

A novel method useful for selective isolation of the C-terminal peptide from a tryptic digestion mixture of a protein has been developed by taking advantage of a unique property of anhydrotrypsin, which has a strong specific affinity for the peptides containing arginine or lysine at their C-termini. Briefly, peptides produced by tryptic digestion of a protein are fractionated by affinity chromatography on a column of immobilized anhydrotrypsin. The C-terminal peptide is recovered in a breakthrough fraction, while the remainders are adsorbed on the column (unless the protein ends in arginine or lysine). The breakthrough fraction is then subjected to reversed-phase high-performance liquid chromatography in order to purify the C-terminal peptide. Using this method, we have successfully isolated the C-terminal peptides from tryptic digests of the sheath protein (gp 18) and the tube protein (gp 19) of bacteriophage T4. The analytical results on these peptides, together with the information on the N-terminal structures of the original proteins and on the nucleotide sequences of genes 18 and 19, allowed us to establish the complete primary structures of the two proteins.     

Affinity chromatography on immobilized hog gastric mucin and ovomucoid. A general method for isolation of lectins

Two affinity adsorbents of general applicability for isolating of plant lectins are presented. The isolation of 30 lectins from 27 plants is described. The method works quickly and is inexpensive. In particular, mixtures of different lectins occurring in some plants may be resolved in one run.     

Affinity chromatography of serum proteins using immobilized lectin from Vicia faba

When human serum is applied to a column of Sepharose-insolubilized lectin from Vicia faba, some serum proteins are bound which can be eluted by means of 0.1 M glucose solution. These proteins are parts of the immunoglobulins IgA, IgG, IgM, and the alpha2-macroglobulin. These particular types of serum protein are bound specifically, due perhaps to some structural variation in the carbohydrate moieties they contain.     

Affinity chromatography of human serum proteins using immobilized lectin from Allomyrina dichotoma

The beta-D-galactoside-specific lectin from Allomyrina dichotoma reacts with serum proteins which contain the corresponding carbohydrate moieties. By affinity chromatography of human serum using the insolubilized lectin coupled to Sepharose, it is possible to fractionate human serum proteins in 2 groups: those which react with the lectin (alpha 1-acid glycoprotein, haptoglobin, etc.) and those which do not (albumin, Gc-globulin, etc.). IgG is the only serum protein that can be found in both groups.     

Separation of peptides on the basis of the difference in positive charge: simultaneous isolation of C-terminal and blocked N-terminal peptides from tryptic digests

The microscale separation of peptides based on the difference in positive charge was examined with tryptic digests of apomyoglobin and calmodulin. By this separation method, C-terminal and blocked N-terminal peptides could be selectively isolated in the same fraction without any chemical modifications. Separated peptides, including internal peptides, were further purified by reversed phase high performance liquid chromatography, and the purified peptides could be directly subjected to sequence and amino acid analyses. The N-terminal peptides of calcium-activated neutral protease were successfully isolated by this method.     

Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding properties from cartilage.

Partially degraded hyaluronate was coupled to AH-Sepharose 4B using carbodiimide. Approximately 1 mg of hyaluronate was incorporated per ml of wet gel. The derivatized gel was used to purify components of the hyaluronate-proteoglycan complex of cartilage. Two link-proteins were isolated from a crude cartilage extract by affinity binding to the gel and eluted with 4 M guanidinium chloride. By the same procedure one link-protein and the globular portion of the proteoglycan monomer were isolated from a trypsin-treated cartilage extract and were separated from each other by subsequent gel chromatography on Sepharose 6B and Sephacryl S-200. The affinity technique was also used for the preparation of these proteins labelled with dansyl groups.     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Affinity chromatography of human alpha-fetoprotein on immobilized estrogens

Human alpha-fetoprotein was isolated from abortive material with the help of affinity chromatography on immobilized estrogens. After butanol extraction from the abortive material human AFP obtained the ability for affinity binding with immobilized estrogens. The addition of estrogens to the extract of isolated AFP preparation and incubation with them did not lower AFP binding with immobilized estrogens during the experiments, using affinity chromatography. A 10% buffered aqueous butanol solution was most optimal for elution. The data obtained can suggest that AFP in biological fluids is bound to estrogens, and butanol extraction deestrogenizes human AFP. The mechanism of AFP binding to estrogens in vivo is, evidently, carried out with the help of specific unknown carrier, as AFP does not bind free estrogens.     

Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Selective isolation and identification of N-terminal blocked peptides from tryptic protein digests.

A method for the easy isolation and direct sequencing of N-terminally blocked peptide in proteins refractory to N-terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the Nalpha-blocked peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N-terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.     

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins

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Affinity chromatography of nicotinamide nucleotide-dependent dehydrogenases on immobilized nucleotide derivatives

A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for `general ligand' affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD⁺ and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD⁺ to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.     

Affinity chromatography of sn-glycerol-3-phosphate dehydrogenase on immobilized NAD

Three types of potential affinity chromatography columns have been examined for the purification of sn-glycerol-4-phosphate dehydrogenase (EC 1.1.1.8) from rabbit tissues. Each column contained nicotinamide adenine dinucleotide (NAD) covalently attached to an agarose matrix with a different mode of attachment for each column. The most effective column was one in which the NAD was linked to the agarose via the C-8 position of the adenine moiety. Release of the bound enzyme from this column was accomplished by elution with NADH or NAD. The enzymes from brain, heart, kidney, muscle and liver were purified using this procedure with nearly quantitative yields and up to a 90-fold purification. The binding capacity and elution profiles were dependent upon pH, ionic strength and temperature. The capacity was lowest at pH 7 and increased at higher and lower values. Increasing ionic strength and higher temperatures decreased the binding capacities.     

Affinity chromatography of nonhistone chromosomal proteins from lymphocytes on DNA-agarose columns

A two step procedure is presented consisting of hydroxyapatite and DNA-agarose chromatography which allows the isolation of nonhistone chromosomal proteins with different affinities towards single stranded DNA. The application of this fractionation scheme to nonhistone chromosomal proteins from bovine lymphocytes is described.     

Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.     

Characterization of proteins and peptides by high-performance liquid chromatography and fluorescence monitoring of their tryptic digests.

Proteins and peptides can be characterized and compared at the subnanomole level by treatment with trypsin followed by high-performance liquid chromatography on reverse-phase partition columns. A fluorescamine monitoring system automatically analyzes a portion of the column effluent while the remainder can be collected for further studies. The method has already been used for characterization of rat β-endorphin and a protein which cross-reacts with antiserum prepared against prolyl hydroxylase.     

Affinity chromatography of bull seminal proteins on mannan-Sepharose

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC-MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 microl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC-MS 9,9,9-D(3)-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring-d(5), 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at -80 degrees C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at -80 degrees C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.