Potential Affinity Labels Research Articles

Synthesis and biological activity of some novel, chemically reactive glucocorticoids

Six chemically reactive derivatives of cortisol were synthesized in order to probe the steroid binding site of glucocorticoid receptors at increasing distances from the C 21 of cortisol. For this purpose, four new affinity label groups were employed, i.e. α-keto-mesylate, α-keto-thiol, thiol acetate and xanthate. The cortisol-21-thiol was prepared by a new synthesis of α-keto thiols. The thiol acetate and xanthate substituents also embody a unique approach to affinity labelling, wherein a small non-steroidal group could be transfered to the receptor in an attempt to obtain functionally active, covalently labelled receptors. The affinities for receptors and the biological activities of the six synthetic steroids were examined in two lines of rat hepatoma tissue culture cells—FU-55 and HTC. The relative affinities of these steroids for receptors did not correlate with the bulk of the steroid substituents. The thiol, thiol acetate and xanthate derivatives were better competitors of [ 3H]dexamethasone binding than the parent steroid cortisol. Interaction of cortisol-thiol acetate and cortisol-xanthate with HTC cell receptors was further examined in a cell-free exchange assay that renders steroid-free receptors incapable of binding the subsequently added [ 3H]-steroid. The behavior of the reactive thiol acetate and xanthate derivatives in this exchange assay, plus their unexpectedly high apparent affinity for receptors, are consistent with the formation of a functionally active, covalently labelled glucocorticoid receptor via transfer affinity labelling. The biological activity of all six steroids in whole cells indicates that these potential affinity label functional groups may be most useful in cell-free studies.

Pyridoxal phosphate as a probe of reovirus transcriptase.

The ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.

Binding specificity of rat alpha-fetoprotein for a series of estrogen derivatives: studies using equilibrium and nonequilibrium binding techniques.

The binding specificity of rat alpha-fetoprotein (AFP) for a series of estrogen derivatives has been investigated and compared to that of the uterine cytosol estrogen receptor. In addition, several equilibrium and nonequilibrium binding techniques have been examined for their appropriateness in studying AFP-estrogen interactions under different experimental conditions. The ratio of association constants (RAC) of various estrogen derivatives (compared to 17 beta-estradiol; RAC = 100%) were determined using a rapid and convenient miniaturized equilibrium assay that utilizes the partitioning properties of Sephadex G-25. AFP has the highest affinity for estrone derivatives and is especially tolerant of small, nonpolar substituents in the D ring; it binds nonsteroidal estrogens and antiestrogens weakly or not at all. Thus, it has a binding specificity quite unlike that of the estrogen receptor. Several potential affinity labels were studied. Of these, 16-diazoestrone, a photoreactive compound, has the highest affinity for AFP (RAC = 121%). Another equilibrium technique, steady state polyacrylamide electrophoresis, effects a clear separation between AFP and albumin. It is superior to conventional polyacrylamide electrophoresis under conditions where dissociation is rapid. Charcoaldextran adsorption also rapidly removes estradiol that is bound to AFP (t1/2 = approximately 10 min at 0 C). The AFP-estradiol complex appears to be stabilized by adsorption to hydroxylapatite, however, so that hydroxylapatite can be employed in a batchwise manner to assay binding to AFP. This is the most appropriate technique when high concentrations of ligand are used. These studies highlight the marked differences in binding specificity between rat AFP and the uterine cytosol estrogen receptor and indicate the techniques most appropriate for binding measurements under different conditions. This work should facilitate the design of affinity labeling reagents for rat AFP as well.

The synthesis of diazo, halo, and sulfoxy bile acid derivatives: Potential affinity labels

Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH leads to COC1 leads to COCHN2 leads to COCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives.

S-(4-Bromo-2,3-dioxobutyl)-CoA: An affinity label for certain enzymes that bind acetyl-CoA

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site( s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μ m) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K i 's (and K m ) were in the range, 30 to 120 μ m. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.

Synthese von Halogenacetyl‐ und Chlorambucil‐Peptiden des Eledoisins, Physalaemins und der Substanz P als potentielle Affinitätsmarker

Synthesis of Halogenacetyl‐ and Chlorambucil Substituted Peptides of Eledoisin, Physalaemin and Substance P for Affinity LabelingPartial sequences of eledoisin have been synthesized in a stepwise manner using Boc‐aminoacid‐2,4,5‐trichlorophenyl esters. These sequences and partial sequences of physalaemin and substance P were acylated via trichloro — or pentachlorophenyl esters and mixed anhydrides of bromo — and iodoacetic acid. Two eledoisin sequences have been prepared which bear a chlorambucil group. A irreversible binding of these potential affinity labels to the isolated guinea pig ileum was not observed.

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.

Reaction of ribulosebisphosphate carboxylase from [formula omitted] with the potential affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate

Under mild conditions, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate rapidly and irreversibly inactivates ribulosebisphosphate carboxylase from Rhodospirillum rubrum . The substrate ribulosebisphosphate protects the enzyme against inactivation. Incorporation of reagent has been quantitated by reduction of the modified carboxylase with [ 3H]NaBH 4. Based on the difference in the levels of incorporation found in the inactivated enzyme as compared with the protected enzyme, loss of enzymic activity results from the modification of about 0.4 residue per catalytic subunit. Analyses of hydrolysates demonstrate that both cysteinyl and lysyl derivatives are present in the inactivated carboxylase; the protected sample contains about the same amount of modified cysteine but little of the modified lysine. Thus, inactivation appears to correlate with derivatization of lysyl residues.

29] Carboxylic-phosphoric anhydrides isosteric with adenine nucleotides

Publisher Summary Adenine nucleotide analogs of this type are of interest as potential affinity labels because (1) they are structurally closely similar to the parent nucleotides, (2) after adsorption to the substrate site they can either acylate or phosphorylate amino acid residues, and (3) it is likely that binding of these analogs to the substrate site will frequently be accompanied by partial neutralization of negative charges on the phosphoryl groups that would increase the reactivity of the mixed anhydride function in the enzyme-bound analogs and thereby tend to increase the specificity of labeling of the substrate site. This chapter describes the synthesis of carboxylic-phosphoric mixed anhydride analogs of AMP (I) and ATP (II) and conditions under which they appear to react covalently at the adenine nucleotide sites of three enzymes.

63] Dimethylpyrazole carboxamidine and related derivatives

Publisher Summary The frequency and great biological importance of divalent metal binding sites on proteins makes them of particular interest as the object of affinity labeling studies. Organic cations of many types interact with metal ion sites in biological materials; the binding of amidine and guanidinium compounds at these sites has been extensively studied. The chapter examines the principles involved in this affinity labeling technique to mitochondria by using a more hydrophobie derivative, 4-phenyl-3,5-dimethylpyrazole-1-carboxyamidine. It details the preparation reagents, preparation of affinity-labeled Pyruvate Kinase, and characterization of 4-Phenyl-3,5-Dimethylpyrazole-1-carboxamidine (PDMPA) as a Potential Affinity Label for Mitochondria. The chapter concludes that several unlabeled guanidinium compounds, including butylbiguanide, phenethylbiguanide, and phenethylguanidine, have been tested as potential binding competitors, hence protective ligands, but none are effective, nor is Ca2+ competitive. Conversely, unlabeled PDMPA does not compete for the nonenergized binding of 45Ca2+. 22.

6] Catalytic competence: A direct criterion for affinity labeling

Publisher Summary Affinity labels or active-site-directed irreversible enzyme inhibitors are designed by combining in the same molecule a moiety that resembles the substrate for an enzyme and a reactive group that will attack some amino acid residue in the protein with the formation of a covalent bond. The success of affinity labeling depends upon the fortuitous presence of a reactive amino acid residue at such a distance from the substrate binding site that the covalently bound substrate like moiety can rest either in the active site or very close to it. This chapter explains the four criteria that have been established for the definition of affinity labels. It examines and provides evidence that the reaction of potential affinity label, 3-bromoacetoxyestrone, with human placental estradiol dehydrogenase and have found that it satisfies the first four criteria for an affinity label. Furthermore, the 17-keto group of this compound offers an opportunity to determine whether, after reaction with the enzyme, the covalently bound steroid is suitably positioned to serve as a substrate in the dehydrogenase reaction.

2] Transition state analogs as potential affinity labeling regents

The principle of transition state affinity requires the development during enzyme catalysis of very powerful forces of attraction between the enzyme and the altered form of the substrate that is present in the transition state. This principle has served as a basis for the synthesis of some very strong reversible enzyme inhibitors, and may also in principle be useful in the design of affinity labeling reagents. The latter possibility remains to be explored, but seems already to be partly substantiated by a few serendipitous examples of inhibitors of glycosidases and proteases. This chapter explains the principle of transition state affinity, methods of examining potential transition of state analogs, possible transition state analogs and multisubstrate analogs and affinity labeling with transition state analogs.

Sulfhydryl analogues of adenosine diphosphate: chemical synthesis and activity as platelet-aggregating agents.

2-Thioadenosine 5'-diphosphate (2-SH ADP), 2,2'-dithiobisadenosine 5'-diphosphate (2,2'-(S-ADP)2), 8-thioadenosine 5' diphosphate (8-SH ADP), and 6-mercaptopurine riboside 5'-disphosphate (6-MPRDP) were synthesized as potential affinity labels for ADP receptors on the blood-platelet membrane. The mean relative activities of these compounds in aggregating human platelets suspended in homologous plasma were 155% (2,2'-(S-ADP)2), 74% (2-SH ADP), 0.65% (8-SH ADP), and 0.08% (6-MPRDP). The mean relative activities against washed platelets were 249% (2,2"-(S-ADP)2) and 115% (2-SH ADP), whereas no aggregation occurred with 8-SH ADP or 6-MPRDP. The last two compounds were found to be weak inhibitors of ADP-induced aggregation. Therefore, thio-substitution at postition 2 followed by oxidation to a disulfide appears to be the most promising approach to further studies of affinity labelling of membrane ADP-receptors.

Photo-affinity labeling of β-D-xylosidase

As part of a comparative study of the structure and function of a group of glycosidases, we are particularly interested in the properties of a P-Dxylosidase (/3-D-xyloside xylohydrolase, EC 3.2.1.37) induced in Bacillus pumilus [l] . In an attempt to obtain further information on the catalytic site of this enzyme, several potential affinity-labels have been prepared [2-41 and tested on this enzyme. This communication reports on the interaction of a photoaffnity label with the fl-D-xylosidase. The choice of a photolytic activatable ligand is founded on practical considerations, the most important being the easy handling of these products, since the reactive species is formed in situ. The advantage of the aromatic azides, used in this work, over other photolabile molecules (e.g. diazoketones), is their thermal and pH stability. On illumination they produce a highly reactive aryl nitrene, which is thought to be able to react with all amino acid residues. It is concluded that p-azidophenyl xylopyranoside acts on the /3-D-xylosidase as a pseudo photo-affinity label, rather than a true photo-affinity label.

Alkylating derivatives of amino acids and peptides. Synthesis of N-maleoylamino acids, [1-(N-maleoylglycyl)cysteinyl]oxytocin. Effects on vasopressin-stimulated water loss from isolated toad bladder.

A method for the preparation of N-maleoylamino acids and esters is reported. These compounds were shown to inhibit both the oxytocin-induced smooth muscle contraction in the isolated rat uterus and the vasopressin-induced water loss from the isolated toad bladder. The inhibitory ability of the maleimides in the toad bladder assay was found to be related to their corresponding partition coefficients by the equation: log 1/C = -0.055 (log P) 2 + 0.227 log P + 3.96. N-Maleoylamino acids can be coupled to peptides to form alkylating reagents which react rapidly with sulfhydryl groups. The synthesis of [1-(N-maleoylglycyl)cysteinyl]oxytocin (3) and [1=(N-maleoyl-11-aminoundecanoyl)cysteinyl]oxytocin (4) as potential affinity labeling reagents is described. These oxytocin analogs were shown to readily react with sulfhydryl-containing compounds; however, neither 3 nor 4 was seen to inhibit in the rat uterus assay at concentrations up to 3 times 10(-5)M. When tested on the mucosal and serosal surfaces of the toad bladder, assay inhibition was seen only on the mucosal surface. These results are discussed with respect to the possible existence of sulfhydryl groups at neurohypophyseal receptors.

Synthesis and characterization of 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate, a potential affinity label for enzymes that bind sugar bisphosphates.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and characterization of 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate, a potential affinity label for enzymes that bind sugar bisphosphatesFred C. HartmanCite this: J. Org. Chem. 1975, 40, 18, 2638–2642Publication Date (Print):September 1, 1975Publication History Published online1 May 2002Published inissue 1 September 1975https://doi.org/10.1021/jo00906a014RIGHTS & PERMISSIONSArticle Views54Altmetric-Citations10LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (742 KB) Get e-Alerts Get e-Alerts

Differences in active center reactivity of trypsin homologs: Specific inactivation of thrombin by nitrophenyl p-amidinophenylmethanesulfonate

In the hope of obtaining a reagent capable of discriminating among trypsin-like enzymes and inactivating one of them, nitrophenyl m- and p-amidinophenylmethanesulfonate were synthesized as potential affinity labels. Both reagents are competitive inhibitors of trypsin, thrombin, plasmin, chymotrypsin, and plasma kallikrein. However, in the case of thrombin alone, the p-isomer achieves a covalent modification irreversibly inactivating the enzyme. The results demonstrate the selectivity possible with active-site-directed reagents even among enzymes with homologous active centers.

Inhibition of α-chymotrypsin by hydroxymethyl analogues of d- and [formula omitted] and N-acetyltryptophan of potential affinity labeling value

d- and l -N- acetyl - and N, O-diacetylphenylalaninol and -tryptophanol are found to be competitive inhibitors of α-chymotrypsin with K 1 values in the range 4.3–65.7 mM. In each case, the N,O-diacetates are better inhibitors than the corresponding hydroxymethyl compound and although hydrogen bonding of hydroxymethyl groups at the nucleophilic site ( n) does contribute to binding, interactions of O-acetate functions with this region are much more significant. The tryptophanol derivatives are more effective by a factor of about 5 than those of phenylalaninol and d enantiomers bind approximately twice as strongly as their l counterparts. The results obtained indicate that phenylalaninol, or preferably tryptophanol, structures of the types studied are suitable for use as alkylating group carriers in affinity labelling experiments designed to probe the more remote amino acid residues of the n-region of the active site. They are also of potential value for X-ray and NMR studies on the differences between the binding of d and l enantiomers by α-chymotrypsin.

Azido compounds as potential affinity labels for glycosidases

Azido compounds as potential affinity labels for glycosidases

The potential of carboxylic-phosphoric mixed anhydrides as specific reagents for enzymic binding sites for alkyl phosphates. Inactivation of an enzyme by an anhydride isosteric with adenosine 5′-phosphate

Adenine nucleotide analogs of this type are of interest as potential affinity labels because (1) they are structurally closely similar to the parent nucleotides, (2) after adsorption to the substrate site they can either acylate or phosphorylate amino acid residues, and (3) it is likely that binding of these analogs to the substrate site will frequently be accompanied by partial neutralization of negative charges on the phosphoryl groups that would increase the reactivity of the mixed anhydride function in the enzyme-bound analogs and thereby tend to increase the specificity of labeling of the substrate site. This chapter describes the synthesis of carboxylic-phosphoric mixed anhydride analogs of AMP (I) and ATP (II) and conditions under which they appear to react covalently at the adenine nucleotide sites of three enzymes.