3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent

A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect protein SH groups on dot blots with sensitivities in the femtomole range. The labeling was very specific for sulfhydryl groups or reduced S-S bonds; proteins lacking free SH groups were unlabeled by this method. Due to the long spacer between the biotinyl group and the reactive maleimide, improved adsorption of biotinylated proteins to avidin columns was achieved. An SH-containing enzyme (beta-galactosidase) was biotinylated with MPB, and the resultant biotinylated enzyme could be used as an efficient histochemical probe. The use of this reagent is recommended to biotinylate proteins which contain nonessential SH groups or which can be easily thiolylated prior to reaction with MPB.     

Biocytin hydrazide--a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology

Biocytin hydrazide (BCHZ), a new, water-soluble, long-chained, biotin-containing hydrazide, was synthesized and used for the selective nonradioactive detection of glycoconjugates. Procedures were developed for labeling glycoconjugates on blots. The method involves either chemical (periodate-induced) or enzymatic (via galactose oxidase) oxidation of glycoconjugates, the resultant aldehyde groups are then labeled with biocytin hydrazide, followed by interaction with an avidin-based enzyme probe. Since the biotin-containing reagent is a relatively small, charged molecule, the primary labeling step may be carried out on intact cells and on membrane preparations as well as on blotted samples. On blots, the labeling pattern was similar for both periodate- and galactose oxidase-induced biotinylation procedures. In contrast, periodate-induced labeling of either erythrocyte membranes or cells (prior to blotting) produced an altered labeling pattern. Combined enzyme-induced biotinylation of membranes or cells resulted in a pattern similar to that observed for the direct staining of blots. Using galactose oxidase on human erythrocyte membranes, the procedure was sensitive enough to selectively label the Band 3 lactosaminoglycoprotein.     

A versatile procedure for the radioionization of proteins and labeling reagents

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.     

A versatile procedure for the radioiodination of proteins and labeling reagents

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.     

Use of streptavidin to detect biotin-containing proteins in plants

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.     

Reactive group-embedded affinity labeling reagent for efficient intracellular protein labeling

Affinity labeling of a target protein is a powerful method for chemical biology studies. However, it is still difficult to label intracellular proteins efficiently in living cells. We propose the novel design strategy of a reactive group-embedded affinity labeling reagent for efficient protein labeling. With FKBP12 as the model target protein, the ligand binding pocket-oriented labeling reagent could label intracellular protein, whereas protein surface-oriented reagent was ineffective for labeling in living cells, partially because of the intracellular protein fluctuation under the macromolecular crowding effects. These results provide new insight for efficient intracellular protein labeling.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.     

Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.     

Reversible fluorescence labeling of amino groups of protein using dansylaminomethylmaleic anhydride

The reversible fluorescence labeling of insulin, catalase and lysozyme has been demonstrated. As a derivatizing reagent, dansylaminomethylmaleic acid (DAM) has been used after investigating the precolumn and precapillary derivatization conditions. This reagent (DAM) reacts with the amino groups of proteins via its anhydride in the presence of a suitable dehydrating reagent, which then could be liberated under mild acidic conditions and the native proteins are regenerated. After the derivatization of insulin, catalase and lysozyme with DAM, no peaks of these native proteins were observed while several peaks of the derivatized proteins due to the multiple labeling were observed. However, after the regeneration, increasing amounts of the native proteins were observed as the regeneration period increased. For the lysozyme, the bacteriolytic activity of the enzyme decreased after the derivatization, and only 0.9% of the activity remained. The activity increases by the regeneration, and 95.6% of the bacteriolytic activity of the native enzyme was observed after a 48-h regeneration at pH 2.5 and 40 degrees C.     

Labeling of membranes from erythrocytes and corn with fluorescamine

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.     

Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril.

The use of the iodinating reagent 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (chloroglycoluril) to selectively label membrane surface proteins was investigated with the following systems: enveloped viruses (Sendai and Newcastle disease viruses), human erythrocytes, and nucleated cells propagated both in suspension (EL-4) and in monolayer culture (BHK-21). Conditions are described for specifically iodinating surface proteins while maintaining full virus integrity or cell viability. Comparison of the chloroglycoluril method with the lactoperoxidase and chloramine-T methods for labeling surface membrane proteins shows that the chloroglycoluril method has a number of advantages: It routinely produces a 3- to 17-fold greater specific radioactivity without sacrificing viral or cellular integrity, it is technically simpler to use, it does not require the addition of extraneous protein to initiate the reaction nor a strong reducing reagent to terminate it. Chloroglycoluril also proved to be an effective substitute for chloramine-T in the nonvectorial labeling of viral and cellular proteins. Membrane protein samples were solubilized with the detergent sodium dodecyl sulfate before iodination or labeled in the presence of high iodide concentrations without prior solubilization. The resulting specific radioactivities generated by the use of chloroglycoluril were equal to or greater than those generated by the chloramine-T method. The effectiveness, simplicity of use, and versatility of chloroglycoluril recommend it as an iodinating reagent for both surface-specific and nonvectorial labeling of membrane systems.     

Isolation of proteins that interact with the signal transduction molecule Dof and identification of a functional domain conserved between Dof and vertebrate BCAP

Dof is a large molecule essential for signal transduction by the two FGF receptors in Drosophila. It contains two ankyrin repeats and a coiled-coil region, but has no other recognisable structural motif. Dof shares these features with its closest vertebrate relatives, the B-cell signalling molecules BCAP and BANK. In addition, this family of proteins shares a region of homology upstream of the ankyrin repeats, which we call the Dof/BCAP/BANK (DBB) motif. We have identified 44 proteins that interact with Dof in a yeast two-hybrid screen. These include the Drosophila FGF-receptor Heartless and Dof itself. We show that the integrity of the DBB motif is required both for Dof and for BCAP to form dimers. Analysis of the interactions between a set of deletion constructs of Dof and the panel of interactors suggests that Dof may adopt different conformations, with a folded conformation stabilized by interactions between the DBB motif and the C-terminal part of the protein.     

Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) is a useful reagent for the affinity labeling of adenine nucleotide binding proteins. We have developed an immunochemical approach to the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of a radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate dehydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots only with proteins that have been labeled previously with the affinity reagent. The degree of immunoreactivity on Western blots correlates well with the extent of covalent modification as shown by studies on the modification and inhibition of the catalytic subunit of cAMP-dependent protein kinase. In crude cellular extracts, numerous proteins can be labeled with FSBA and then detected by using this approach. The labeling and subsequent detection of these proteins can be blocked by including an excess of MgATP, which competes with FSBA for nucleotide-binding sites. The labeling of specific proteins in crude mixtures is saturable, as shown by labeling studies of p56lck, a protein-tyrosine kinase that is abundantly expressed in membranes from the T lymphoma cell line LSTRA.     

Protein analysis on two-dimensional polyacrylamide gels in the femtogram range: use of a new sulfur-labeling reagent

An ultrasensitive staining procedure for two-dimensional polyacrylamide gels of protein homogenates has been developed. It combines the use of ultrathin gels and the labeling of proteins by a 35S-labeling reagent.     

Site-selective lysine modification of native proteins and peptides via kinetically controlled labeling

The site-selective modification of the proteins RNase A, lysozyme C, and the peptide hormone somatostatin is presented via a kinetically controlled labeling approach. A single lysine residue on the surface of these biomolecules reacts with an activated biotinylation reagent at mild conditions, physiological pH, and at RT in a high yield of over 90%. In addition, fast reaction speed, quick and easy purification, as well as low reaction temperatures are particularly attractive for labeling sensitive peptides and proteins. Furthermore, the multifunctional bioorthogonal bioconjugation reagent (19) has been achieved allowing the site-selective incorporation of a single ethynyl group. The introduced ethynyl group is accessible for, e.g., click chemistry as demonstrated by the reaction of RNase A with azidocoumarin. The approach reported herein is fast, less labor-intensive and minimizes the risk for protein misfolding. Kinetically controlled labeling offers a high potential for addressing a broad range of native proteins and peptides in a site-selective fashion and complements the portfolio of recombinant techniques or chemoenzymatic approaches.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate, a new carbene generating reagent for probing hydrophobic membrane domains

A new precursor of a lipophilic photolabel, 2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate (NADIT) has been synthesized. The suitability of the reagent for labeling the hydrophobic core of membranes is demonstrated by studying its reactivity in chromatophores of Rhodospirillum rubrum G-9+. The label binds preferentially to the phospholipids and intrinsic membrane proteins. In isolated reaction centers treated with NADIT the hydrophobic subunits M and L are more labeled than the H subunit. The high reactivity, dark stability and ease of synthesis favors this very lipophilic reagent to identify the intrinsic hydrophobic sections of membrane proteins.     

Fluorescent labels for proteomics and genomics

Fluorescent labeling reagents are an essential component of a huge industry built on sensitive fluorescence detection. This technology has grown over 30 years and is in some ways mature. Excellent labeling reagents with close to maximum theoretical brightness are available in many different colors. Large fluorescent proteins like phycobiliproteins are also widely used that are exceedingly bright. Other fluorescent proteins like the GFP family can be obtained for creating genetically encoded protein labels in living cells. A new 'solid state' quantum dot technology is being exploited for large-scale multiparameter labeling. This technology provides the 'ultimate' photostable labeling reagent. Still, there are advances to be made. Not available is the ultimate tool kit of low molecular weight, strongly light absorbing, photostable labels with narrow emission bands ranging from the UV to the IR.     

Outer membrane of Salmonella typhimurium. Identification of proteins exposed on cell surface.

Proteins exposed on the outer surface of the outer membrane of Salmonella typhimurium were identified by reacting intact cells with a covalent labeling reagent. Since the outer membrane permitted the free diffusion of small hydrophilic molecules, we used a macromolecular reagent, CNBr-activated dextran, as the non-penetrating labeling agent. We also used a mutant producing a lipopolysaccharide with a very short (i.e. hexasaccharide) carbohydrate chain, in order to avoid steric hindrance by the carbohydrates on membrane surface. Results showed that out of the four "major" proteins of molecular weight around 35 000, three were exposed, and that at least six other proteins were also exposed on cell surface. Only two or three outer membrane proteins consistently did not react with the reagent in intact cells.