Reaction of peptide 89-169 of bovine myelin basic protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine.

The C-terminal half of the bovine myelin basic protein, peptide 89-169, was treated with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine], and the products were isolated by repeated gel filtration through Sephadex G-50. They consisted of uncleaved peptide 89-169 in which approximately 30% of the tyrosine had been monobrominated and the tryptophan converted to oxindolealanine, peptide 116-169 modified by partial bromination (30%) of the tyrosine, and two chromatographic forms of peptide 89-115. The major form contained the lactone of dioxindolealanine at the C terminus; the minor form contained the uncyclized oxidation product. Each form of peptide 89-115 was resolved into several components by electrophoresis in polyacrylamide gels (10%, w/w) containing 1 M acetic acid and 8 M urea. The presence of three of these components could be explained by partial deamidation of Asn-91 and Gln-102. Studies on the oxidation of tryptophan-containing model peptides by BNPS-skatole indicated that the reaction can also include partial bromination of the dioxindole and its lactone and partial cleavage at the amino peptide bond of the tryptophan.     

Development of an acid hydrolysis method with high recoveries of tryptophan and cysteine for microquantities of protein.

High recoveries of tryptophan and cysteine were achieved by 12.5 min of hydrolysis with mercaptoethanesulfonic acid vapor. Proteins (1-100 micrograms) were modified by vapor-phase S-pyridylethylation before hydrolysis. The modified proteins were hydrolyzed with the vapor of 2.5 M mercaptoethanesulfonic acid at 176 degrees C. This method promoted efficient hydrolysis of the peptide bonds in proteins and resulted in high recoveries of both tryptophan and cysteine, of 90% or greater, in addition to the other amino acids.     

UV Raman studies of peptide conformation demonstrate that betanova does not cooperatively unfold.

We used UV resonance Raman spectroscopy (UVRR) excited within the peptide bond pi --> pi* electronic transitions and within the aromatic amino acid pi --> pi* electronic transitions to examine the temperature dependence of the solution conformation of betanova, a 20-residue beta-sheet polypeptide [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256]. The 206.5 nm excited UVRR enhances the amide vibrations and demonstrates that betanova has a predominantly beta-sheet structure between 5 and 82 degrees C. The 229 nm excited UVRR, which probes the tyrosine and tryptophan side chain vibrations, shows an increase in the solvent exposure of the tryptophan side chains as the temperature is increased. Our results are consistent with the existence of an intermediate state similar to that calculated by Bursulaya and Brooks [Bursulaya, B. D., and Brooks, C. L. (1999) J. Am. Chem. Soc. 121, 9947-9951] and exclude the previously proposed two-state cooperative folding mechanism. Betanova's structure appears to be molten globule over the 3-82 degrees C temperature range of our study.     

High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid.

A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.     

The effect of copper on frog skin: the role of sulphydryl groups.

1. Cu2+ at a concentration of 10-4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc). 2. This effect was studied in skins of Rana temporaria adapted to cold,(5 degrees C) and room temperature (20 degrees C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+. 5. 5,5' -dithiobis (2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.     

Purification and characterization of the aromatic desulfinase, 2-(2'-hydroxyphenyl)benzenesulfinate desulfinase

2-(2(')-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon-sulfur bond of 2-(2(')-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS-PAGE. The pI of HPBS desulfinase is 5.6, the temperature optimum is 35 degrees C, and the pH optimum is 7.0. HPBS desulfinase has a K(m) of 0.90+/-0.15 microM and a k(cat) of 1.3+/-0.07 min(-1). Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu(2+) and Zn(2+), while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.     

Oxidative inactivation of the lymphoid tyrosine phosphatase mediated by both general and active site directed NO donors

Oxidative modification of protein tyrosine phosphatases (PTPs) has recently been recognized as an important regulatory mechanism in biological systems. Reported herein is the oxidative inactivation of the lymphoid tyrosine phosphatase (LYP) with both the general nitrosating reagent sodium nitroprusside (SNP) and also a novel peptide-based nitrosating reagent, Ac-ARLIEDNE(HcyNO)TAREG-NH₂, where HcyNO = S-nitrosohomocysteine. The SNP oxidatively inactivated LYP with a k_inact of 0.383 per min and a K_I of 27.4 μM and mixed-type inactivation kinetics. The peptide was a competitive LYP inactivator with a k_inact of 0.0472 per min and a K_I of 7.00 μM. LYP nitrosation by SNP was characterized by the addition of several NO moieties to the enzyme, while oxidation of LYP by the peptide did not result in the formation of a LYP-NO adduct. We propose that general NO donors promiscuously nitrosate any free cysteine residue while the active-site directed peptide selectively oxidizes the catalytic cysteine residue, resulting in the formation of a disulfide bond between the catalytic cysteine residue and a second cysteine in the active site.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Metabolism of alanylalanyl-S-[N-(2-thioethyl)]aminopyridine-2, 6-dicarboxylic acid]cysteine by suspensions of Escherichia coli

The attachment of 4-[N-2-(mercaptoethyl)]aminopyridine-2,6-dicarboxylic acid (MEPDA) to AlaAlaCys through a disulfide bond to the cysteine residue has been described (Boehm, J. C., Kingsbury, W. D., Perry, D., and Gilvarg, C. (1983) J. Biol. Chem. 258, 14850-14855). The peptide disulfide showed enhanced growth inhibitory properties in Escherichia coli compared to the free sulfhydryl compound. Genetic evidence was presented to show that this side chain-modified peptide utilizes the oligopeptide transport system to gain entry to the cell. Following transport of the peptide, MEPDA is liberated by disulfide exchange reactions with sulfhydryl-containing components of the cell pool. In this paper, we examine in more detail the metabolism of this peptide. Using gel filtration chromatography to examine filtrates from cell suspensions incubated with the peptide, it was shown that loss of the peptide from the medium is accompanied by a corresponding increase in a component having the properties of MEPDA. The release of sulfhydryl groups from the peptide by cell suspensions could be monitored by Ellman's reagent and was found to be dependent upon peptide transport. Following cleavage of the disulfide bond, MEPDA is able to cross the cytoplasmic membrane and exit from the cell as a relatively lipophilic uncharged metal chelate.     

Catalytic Properties of Tryptophanase from Proteus rettgeri

Formation of sclerotia in a strain of Sclerotinia libertiana Fuckel using Czapek-Dox agar medium was highest at pH 4.0~6.0 and at 22~25°C. The response was better under darkness than under light. However, when a potato-extract medium was used the optimum temperature range extended, and even at 15~27°C mature sclerotia formed. The addition of indole-3-acetic acid (IAA) to the Czapek-Dox medium containing riboflavine stimulated the formation of sclerotia under fluorescent light. Though iodoacetic acid, a ?SH reagent, also had a stimulatory effect, cysteine had little inhibitory effect on sclerotial formation. Formation was markedly inhibited by p-aminobenzoic acid (PABA), especially in the presence of tyrosine or tryptophan, and excess ammonium salts in the medium also produced an inhibitory effect. It was assumed that accumulation of an intermediary metabolite with high reactivity with ?SH groups was necessary for sclerotial formation, but PABA and excess ammonium salts inhibited the accumulation.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.     

Estimation of peptide concentration by a modified bicinchoninic acid assay

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.     

Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.     

Use of N-chlorosuccinimide/urea for the selective cleavage of tryptophanyl peptide bonds in proteins. Cytochrome c.

The conditions and utility of the N-chlorosuccinimide/urea (NCS/urea) reagent for the selective cleavage of tryptophanyl peptide bonds in proteins is demonstrated with cytochrome c. At low concentrations of NCS/urea the oxidation of thioether side chains in cytochrome c is the predominant reaction. Methionyl residues are oxidized to sulfoxide and the heme-thioether bridge is partially cleaved. At 10-fold excess of NCS/urea reagent, cleavage of the tryptophanyl peptide bond is optimal at approximately 50% yield in several species of cytochrome c studied. Analytical data on isolated horse cytochrome c peptide fragments demonstrate lack of modification and cleavage at tyrosyl and histidyl residues. However, at high concentrations of NCS/urea reagent (30-fold) unexpected conversions of methionine to sulfone and cysteine to cysteic acid in intact proteins are observed. This is in contradistinction to the absence of sulfone in NCS/urea-reacted amino acid mixtures. The mechanisms of halogenation and cleavage by N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide are discussed. It is porposed that the selectivity with respect to halogenation by N-chlorosuccinimide is due to the insignificant participation of molecular chlorine in the NCS/urea reaction. A mechanism of halogenation and cleavage by NCS at tryptophan is also offered.     

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.     

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.     

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

Interference by lipids in the determination of protein using bicinchoninic acid

Bicinchoninic acid forms the basis of an analytical method for the determination of protein. The reagent produces a purple complex with cuprous ion (Cu+) in an alkaline environment and is the basis for the monitoring of cuprous ions produced in the reactions of proteins with alkaline Cu2+. This method of protein determination was reported to have greater tolerance to many commonly encountered interfering compounds, when compared to the Lowry technique. However, we have found the bicinchoninic acid technique to produce erroneously high values for protein when common membrane phospholipids were included in the assay. Phospholipids in the presence of bicinchoninic acid produced an absorbance peak similar to that produced by protein. This absorbance was linear with concentration, however, the slope varied for individual phospholipids. The combined absorption of phospholipid and protein was not strictly additive. The results indicate that the presence of appreciable quantities of lipid in samples can cause significant error in the analysis of protein by the bicinchoninic acid procedure.