A simplified procedure to determine tryptophan residues in proteins.

A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, 2-hydroxy-5-nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 m urea at acid pH; incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mg/ml of the protein at 280 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

考马斯亮蓝显色液组分对蛋白质测定的影响

考马斯亮蓝（CBB）显色法测定蛋白质含量的主要缺点之一是线性关系差．通过研究显色液组分对线性关系的影响,发现显色液H⁺浓度是影响线性关系的主要因素,并提出了一个新的显色液配方来改善考马斯亮蓝蛋白质测定法的线性关系．     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

Potential interference of hydrogen peroxide in the 2,2'-bicinchoninic acid protein assay

At a concentration of 20-800 nmol/0.1 ml hydrogen peroxide instantly reacts with a 2,2'-bicinchoninic acid copper color reagent. It also reacts with a reformulated reagent at pH 7 but the color develops less rapidly. While the effect may interfere with protein estimations at alkaline pH, the effect of pH 7 may be used to determine the possible extent of hydrogen peroxide interference after treatment with catalase.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Estimation of protein sulfhydryl groups with 5,5'-(35S) dithio-bis(2-nitrobenzoate)

³⁵S-Labeled dithio-bis(2-nitrobenzoate) has been prepared and its usefulness for the measurement of protein sulfhydryl groups has been investigated. The technique is at least 20 times more sensitive than when dithio-bis(2-nitrobenzoate) is used colorimetrically. The adduct formed between the reagent and sulfhydryl groups is stable between pH 3–9 which should enable use of the reagent for the tagging of sulfhydryl-containing proteins in a wide variety of biochemical procedures. Removal of the adduct from a protein is achieved simply by treatment with a thiol reagent, with concomitant restoration of enzymic activity of the protein. The procedure has been successfully tested on alcohol dehydrogenase and mammalian ribosomes.     

Intermolecular cross-linking of a protein crystal--acid protease from Endothia parasitica--in 2.7 M ammonium sulfate solution

A workable procedure for cross-linking of a protein crystal - acid protease from Endothia parasitica - in 2.7 M ammonium sulfate solution with bifunctional reagent glutaraldehyde has been found. Using the cross-linking technique, we have made one good quality heavy atom derivative of uranyl into three or even more isomorphous derivatives for this particular protein.     

Rapid solid phase isolation of 20 specific tRNAs from Escherichia coli.

A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is sufficient for the isolation of 200 mg of a specific tRNA. The procedure requires a bifunctional reagent, one moiety of which (--SO2Cl) reacts with the amino acid on the aminoacylated tRNA, the other, with the --SH group on the resin. Thus, only the desired tRNA species is bound to the resin; any of the other tRNAs in the filtrate can be isolated in another cycle. Raising the pH results in deacylation and release from the resin of the desired tRNA species. For tRNA Cys, it is necessary to block the --SH of cysteine prior to reaction with the bifunctional reagent. Side reactions involving the bifunctional reagent. Side reactions involving the bifunctional reagent and tRNA are either easily reversible or negligible (less than 0.01%).     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.     

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

Diacetyl for Blocking the Histochemical Reaction for Arginine

Two different histochemical methods for blocking the guanidinium group of arginine in formalin fixed, paraffin embedded material are reported here. One procedure uses diacetyl with short incubation times and high pH, the other uses the trimer of the diacetyl reagent and requires longer incubation times but moderate pH. The diacetyl reagent is recommended despite its high pH because the preparation of the diacetyl trimer is laborious and time-consuming.     

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.     

Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change

Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium [Welsch, D. J., & Nelsestuen, G. L. (1988) Biochemistry (second of three papers in this issue)] while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 degrees C or with 0.2 M hydroxylamine at 50 degrees C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. The Lys-97 in this peptide was acetylated but did not contain radiolabel. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. Aglycofragment 1, produced by treatment of fragment 1 with HF, was radiolabeled by this procedure; peptide 94-111 was isolated and was further digested with protease. The major radiolabeled product contained Asn101-Ser102 along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: identification and binding directions of major protein complexes

Rat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-aroylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 A, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ---- L13a, L4 ---- L18, L4 ---- L18a, L4 ---- L26, L6 ---- L29, L14 ---- L13a, L21 ---- L18a and L27 ---- L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6' is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.     

Histochemical detection of alpha-D-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-alpha-D-glucoside

5-Br-4-Cl-3-Indoxyl-alpha-D-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fuchsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid alpha-D-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane alpha-D-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane alpha-D-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid alpha-D-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal alpha-D-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.