A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

Metallocyclopeptide complexes with MII(S.Cys)4 chromophores

The tetracysteinyl peptide cyclo[Lys1,12](Gln-Cys-Gly-Val-Cys-Gly-Lys-Cys-Ile-Ala-Cys-Lys) ([symbol: see text] L(Cys.SH)4) was synthesized by solid-phase methods using an Fmoc/t-Bu/allyl strategy on a PAL-PEG-PS support. The formation of the 1:1 complexes with M = Fe2+, Co2+, and Ni2+ was observed by spectrophotometric monitoring of reactions in aqueous solution at pH 7.5. Size exclusion chromatography indicated that the peptide is a monomer and the complexes are dimers [M2([symbol: see text]L(Cys.S)4)2] in aqueous buffer at pH 7.5. Cobalt and nickel K-edge X-ray absorption data and EXAFS analysis of [Co2([symbol: see text] L(Cys.S)4)2] and [Ni2([symbol: see text] L(Cys.S)4)2] as lyophilized solids are reported. Derived bond distances are Co-S = 2.30 A and Ni-S = 2.21 A. From the collective results provided by absorption spectra, K-edges, EXAFS, and bond length comparisons with known structures, it is shown that [Fe2([symbol: see text] L(Cys.S)4)2] and [Co2([symbol: see text] L(Cys.S)4)2] possess distorted tetrahedral structures and [Ni2([symbol: see text] L(Cys.S)4)2] has distorted square planar stereochemistry. The Co(II) chromophore is particularly distinctive of the assigned structure, displaying three components of the parent tetrahedral ligand field transition 4A2-->4T1(P) (610, 685, 740 nm). The observed structures conform to the intrinsic stereochemical preferences of the metal ions. Structures for the binuclear complexes are suggested. These are the first characterized metal complexes of a cysteinyl cyclopeptide and among the few well-documented complexes of synthetic cyclopeptides. This study is a desirable first step in the design of cyclic peptides for the binding of mononuclear and polynuclear metal centers.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Enhancement of thermostability of the Luciola mingrelica firefly luciferase by site-directed mutagenesis of nonconservative cysteine residues Cys62 and Cys146

Mutant forms of the firefly (Luciola mingrela) luciferase with point mutations Cys62Ser and Cys146Ser were obtained by site-directed mutagenesis. The mutations did not affect the catalytic activity and fluorescence spectra of the enzyme. The rate constants of the fast (k ₁) and slow (k ₂) stages of thermoinactivation of the wild-type and mutant enzymes were determined at 37°C in the absence and presence of 12 mM dithiothreitol (DTT). The thermostability of the mutant forms of luciferase increased several times compared to the wild-type enzyme. In the presence of DTT, k ₂ of the wild-type enzyme decreased three times whereas neither k ₁ nor k ₂ of the mutant forms changed. It was concluded that amino acid residues Cys62 and Cys146 play a major role in luciferase inactivation and that their substitution with Ser stabilizes the enzyme.     

Gemini surfactants from natural amino acids

In this review, we report the most important contributions in the structure, synthesis, physicochemical (surface adsorption, aggregation and phase behaviour) and biological properties (toxicity, antimicrobial activity and biodegradation) of Gemini natural amino acid-based surfactants, and some potential applications, with an emphasis on the use of these surfactants as non-viral delivery system agents. Gemini surfactants derived from basic (Arg, Lys), neutral (Ser, Ala, Sar), acid (Asp) and sulphur containing amino acids (Cys) as polar head groups, and Geminis with amino acids/peptides in the spacer chain are reviewed.     

MC1R Gene Polymorphism Affects Skin Color and Phenotypic Features Related to Sun Sensitivity in a Population of French Adult Women

The melanocortin-1 receptor ( MC1R ) gene is known to play a major role in skin and hair pigmentation and to be highly polymorphic in Caucasians. This study was performed to investigate the relationships between MC1R gene polymorphisms and skin color in a large sample of French middle-aged Caucasian women. The codons 60 to 265 and the codon 294 of the MC1R gene were sequenced in 488 women. The skin color was measured on the inner side of the forearm using a spectrophotometric instrument. Fifteen variants were identified: Arg151Cys, Arg160Trp, Arg142His, Asp294His, Ile155Thr, Asp84Glu, Val60Leu, Val92Met, Arg163Gln, Ser83Pro, Thr95Met, Pro256Ser, Val265Ile, Ala166Ala and Gln233Gln. Women carrying Arg151Cys, Asp294His, Arg160Trp and Asp84Glu variants had a significantly higher reflectance in the red region, which indicates a lower level of functional melanin. This association was the most pronounced for women carrying Asp84Glu. In contrast, no significant difference was observed for other variants. Moreover, associations between MC1R polymorphisms and the risks of experiencing sunburn and of having freckles were found independently of skin color. Our findings support the hypothesis that MC1R polymorphisms do not necessarily alter the skin color but should sensitize the skin to UV-induced DNA damage.     

氨基酸功能化磷钨酸盐的制备及其催化氧化脱硫性能

以L-丙氨酸和磷钨酸为原料,合成氨基酸功能化磷钨杂多酸盐([Ala]3PW),并采用XRD、FT-IR对其结构进行表征。以[Ala]3PW为催化剂,过氧化氢作为氧化剂,甲基咪唑四氟硼酸盐([HMIM]BF4)为萃取剂氧化萃取一体法脱除模拟油中的二苯并噻吩(DBT)。考察了反应温度、催化剂加入量、O/S物质的量比、萃取剂加入量、硫化物类型等因素对脱硫效率的影响。结果表明,在模拟油为10mL、[Ala]3PW=0.04g、O和S的物质的量比为6、V([HMIM]BF4)/V(oil)=0.6、反应温度50℃、反应时间180min的条件下,DBT的转化率可达到98.2%。催化剂循环使用4次活性没有明显的降低。     

Separation of oxidized human growth hormone variants by reversed-phase high-performance liquid chromatography. Effect of mobile phase pH and organic modifier.

Human growth hormone (hGH), a polypeptide of 191 amino acids, contains three methionine residues, two of which are susceptible to oxidation by both chemical and photochemical means (Met14 and Met125). To date, no method has existed for resolving the various mono- and di-oxidation products. We report on the resolution of these oxidized variants and native hGH at weakly (pH 3.5) acidic pH. Although all of the oxidized species can be resolved at pH 3.5, use of low pH and neutral pH mobile phases confer some advantages. For example, the Met14 oxidized variant (MetSO-14) and native hGH are best resolved at neutral pH and the mono-oxidized variants (MetSO-125 and MetSO-14) are best resolved at low pH. The effect of organic modifier on the separation of the oxidized variants was also evaluated. 1-Propanol was more effective than acetonitrile in the separation of MetSO-14 and native hGH while acetonitrile was more effective than 1-propanol in the separation of MetSO-125 and MetSO-14. In summary, mobile phase pH and organic modifier were shown to be important parameters in the separation of oxidized hGH variants.     

Usage of nonporous polymeric particles for reversed-phase high-performance liquid chromatography of oxidized and deamidated forms of recombinant human growth hormone

In this work, a C(18) reversed-phase column with nonporous polymeric 2.5- micro m particles is utilized to initially test the analysis of oxidized and deamidated human growth hormone (hGH). Phosphate buffer (pH 7.5) with 24% 1-propanol was used for elution. This quick method (analysis time is 20 min) gave a selectivity, as judged by the number of detected peaks, and resolution of hGH variants that is better than many methods in which porous silica particle columns are used. Only mixtures of oxidized and deamidated hGH are analyzed, and no characterization of the peaks is performed. The results indicate that C(18) nonporous polymeric column material is a promising alternative for the chromatographic separation of several hGH variants.     

Spectroscopic characterizations of bridging cysteine ligand variants of an engineered Cu2(Scys)2 CuA azurin

Bridging cysteine ligands of the Cu(A) center in an engineered Cu(A) azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser Cu(A) azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pK(a) of the transition is 6.0. A type 1 copper EPR spectrum with A(||) = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A(||) = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.     

Study on physicochemical properties of ionic liquids based on alanine [Cnmim][Ala] (N=2,3,4,5,6)

According to Fukumoto's method, a new series of ionic liquids (ILs) based on alanine, [Cnmim][Ala] ( n=2,3,4,5,6), which comprise 1-alkyl-3-methylimidazolium cation ([Cnmim](+)) and alanine anions ([Ala] (-)), were prepared and characterized. In terms of standard addition method, the density and surface tension of amino acid ILs [Cnmim][Ala] (1-alkyl-3-methylimidazolium alpha-aminopropionic acid salt) were measured in the temperature range 293.15-343.15+/-0.05 K. The volume and surface properties of the ILs [Cnmim][Ala] were discussed. A new method of determining parachor of ionic compound was proposed and was applied to estimate the physicochemical properties of amino acid ionic liquids (AAILs): molecular volume, surface tension, molar enthalpy of vaporization, and thermal expansion coefficient. In comparison with Deetlefs's method of using neutral parachor contribution, the method proposed in this work makes smaller error in estimating properties of AAILs.     

An efficient expression of human growth hormone (hGH) in the milk of transgenic mice using rat Β-casein/hGH fusion genes

In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3′ flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat Β-casein gene promotor. The 3′ flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat Β-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 Μg/mL vs 0.7-2 Μg/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 ±620 Μg/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogenous mouse Β-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat Β-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue-and stage-specific manner in the transgenic mice and the 3′ flanking sequences of hGH gene had an important role for the efficient expression.     

Tracking growth hormone abuse in sport: A comparison of distinct isoform-based assays

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22 kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20 kDa hGH isoforms and also a ratio 22/20 kDa is derived.     

Metal cation dependence of interactions with amino acids: bond energies of Cs+ to Gly, Pro, Ser, Thr, and Cys

The interactions of cesium cations with five amino acids (AA) including glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), and cysteine (Cys) are examined in detail. Experimentally, the bond dissociation energies (BDEs) are determined using threshold collision-induced dissociation of the Cs(+)(AA) complexes with xenon in a guided ion beam tandem mass spectrometer. Analyses of the energy-dependent cross sections include consideration of unimolecular decay rates, internal energy of the reactant ions, and multiple ion-neutral collisions. Bond dissociation energies (0 K) of 93.3 ± 2.5, 107.9 ± 4.6, 102.3 ± 4.1, 105.4 ± 4.3, and 96.8 ± 4.2 kJ/mol are determined for complexes of Cs(+) with Gly, Pro, Ser, Thr, and Cys, respectively. Quantum chemical calculations are conducted at the B3LYP, B3P86, MP2(full), and M06 levels of theory with geometries and zero-point energies calculated at the B3LYP level using both HW*/6-311+G(2d,2p) and def2-TZVPPD basis sets. Results obtained using the former basis sets are systematically low compared to the experimental bond energies, whereas the latter basis sets show good agreement. For Cs(+)(Gly), theory predicts the ground-state conformer has the cesium cation binding to the carbonyl group of the carboxylic acid. For Cs(+)(Pro), the secondary nitrogen accepts the carboxylic acid hydrogen to form the zwitterionic structure, and the metal cation binds to both oxygens. Cs(+)(Ser), Cs(+)(Thr), and Cs(+)(Cys) are found to have tridentate binding at the MP2(full) level, whereas the density functional approaches slightly prefer bidentate binding of Cs(+) at the carboxylic acid moiety. Comparison of these results to those for the smaller alkali cations provides insight into the trends in binding affinities and structures associated with metal cation variations.     

Radioiodination of recombinant human growth hormone and its use in radioimmunoassay

Radioimmunoassay (RIA) of human growth hormone (hGH) using¹²⁵I-labeled tracer prepared from DNA recombinant hGH (r-hGH) and characterization of the tracer in the assay system are described. The radioiodination of r-hGH resulted in high yield of immunoreactive tracer. The immunoreactive fraction could be purified by gel-filtration on sephadex G-75. The quality of radioiodinated tracer of r-hGH has been found to be same as that of the tracer obtained from pituitary hGH (p-hGH) with respect to immunoreactivity, assay sensitivity and RIA standard curve parameters.     

Cross-metathesis and ring-closing metathesis reactions of amino acid-based substrates

Olefin tethers of variable length, introduced into a natural amino acid (side-chain of Ser, Cys; N-terminus of Arg; C-terminus of Phe and Tic; and in both the side-chain and either the N- or C-terminus of Ser, Cys and Tyr), undergo metathesis on treatment with Grubbs' second generation catalyst. Side-chain linked dimers of Ser, Cys and Tyr were obtained by cross-metathesis, while olefin installation at the N- and C-terminus led to dimers of Arg and Phe (or Tic), respectively. Ring-closing metathesis of the doubly alkenylated derivatives of Ser, Cys and Tyr gave 12-, 20- and 24-membered macrocycles.     

Radical-cationic gaseous amino acids: a theoretical study

Three major forms of gaseous radical-cationic amino acids (RCAAs), keto (COOH), enolic (C(OH)OH), and zwitterionic (COO(-)), as well as their tautomers, are examined for aliphatic Ala(.+), Pro(.+), and Ser(.+), sulfur-containing Cys(.+), aromatic Trp(.+), Tyr(.+), and Phe(.+), and basic His(.+). The hybrid B3LYP exchange-correlation functional with various basis sets along with the highly correlated CCSD(T) method is used. For all RCAAs considered, the main stabilizing factor is spin delocalization; for His(.+), protonation of the basic side chain is equally important. Minor stabilizing factors are hydrogen bonding and 3e-2c interactions. An efficient spin delocalization along the N-C(alpha)-C(O-)O moiety occurs upon H-transfer from C(alpha) to the carboxylic group to yield the captodative enolic form, which is the lowest-energy isomer for Ala(.+), Pro(.+), Ser(.+), Cys(.+), Tyr(.+), and Phe(.+). This H-transfer occurs in a single step as a 1,3-shift through the sigma-system. For His(.+), the lowest-energy isomer is formed upon H-transfer from C(alpha) to the basic side chain, which results in a keto form, with spin delocalized along the N-C(alpha)-C=O fragment. Trp(.+) is the only RCAA that favors spin delocalization over an aromatic system given the low ionization energy of indole. The lowest-energy isomer of Trp(.+) is a keto form, with no H-transfer.     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53. Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state [tetramer, dimer, or equilibrium mixture of both] of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met [tetramer] > Ser Asp, His, Gin, > Glu, Lys [dimer], whereas the experimental results showed the following order: Met [tetramer] > Ser > Gln > His, Lys > Asp, Glu [dimers]. For residue 344, the calculated trend was Leu [tetramer] > Ala > Arg, Gln, Lys [dimer], and the experimental trend was Leu [tetramer] > Ala, Arg, Gln, Lys [dimer]. The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer-dimer interface. This initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.