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T W Thannhauser

Publications and source records attributed to T W Thannhauser.

12 recordsLinked to original sources
PubMed1998

Determination of the cysteine and cystine content of proteins by amino acid analysis: application to the characterization of disulfide-coupled folding intermediates.

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.

Cysteamine↗
PubMed1997

Kinetic studies of the regeneration of recombinant hirudin variant 1 with oxidized and reduced dithiothreitol.

The regeneration of native recombinant hirudin variant 1 (rHV1) from the reduced unfolded form to the fully oxidized native state has been carried out with mixtures of oxidized and reduced dithiothreitol at pH 8.3 and 12 degrees C. The regeneration reaction was quenched at various times by the addition of 2-aminoethyl methanethiosulfonate to block unreacted sulfhydryl groups. The quenched protein-folding intermediates were fractionated by both capillary electrophoresis and a combination of anion exchange and reverse phase HPLC and characterized by mass spectrometry, amino acid analysis, and disulfide analysis. These intermediates (before quenching) were found to interconvert rapidly so as to achieve a steady-state distribution early in the regeneration process. The experimental data were fitted to a steady-state kinetic scheme. The analysis reveals that the rate-determining step in the regeneration of rHV1 with oxidized and reduced dithiothreitol involves the oxidation of one or more two-disulfide-containing species, most likely those already containing two native disulfide bonds. This regeneration mechanism is different from one that has been proposed by Chatrenet and Chang [(1993) J. Biol. Chem. 268, 20988]. The differences are discussed, and possible explanations for the differences are presented.

Dithiothreitol↗
PubMed1996

State of aggregation of recombinant hirudin in solution under physiological conditions.

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15 N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight MW determined by sedimentation equilibrium was found to be 6914 +/- 76 Da compared to 6964 Da expected from the amino acid sequence. The MZ/MW ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M,) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991), Eur. J. Biochem. 202, 67-73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konno et al. (1988), Arch. Biochem. Biophys. 267, 158-166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.

Animals↗
PubMed1995

Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry.

Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (< 50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (1o(5)) resolving power makes possible detection of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76-mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 50-mer DNA is characterized with < 10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-stranded DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da.

Base Sequence↗
PubMed1985

Reversible blocking of half-cystine residues of proteins and an irreversible specific deamidation of asparagine-67 of S-sulforibonuclease under mild conditions.

For use in protein-folding studies, a rapid procedure for the preparation of octa-S-sulforibonuclease A (SO3-RNase A) with 2-nitro-5-(sulfothio)benzoate is described. The modification is specific for thiols and disulfide bonds. The modified protein was characterized and found to be enzymatically inactive and predominantly conformationally disordered. In the absence of thiols, the modified sulfhydryl groups were found to be stable over the pH range of 2-9. However, when the modified protein is incubated at neutral to slightly alkaline conditions for prolonged periods of time or at elevated temperatures, it undergoes a further (irreversible) modification that decreases its net charge at pH 8.0. Evidence is presented that demonstrates that this additional modification is due to the specific deamidation of asparagine-67. When incubated with an excess of reduced and oxidized glutathiones for 24 h at pH 8.2 and 25 degrees C, the reversible sulfo blocking group was removed, and essentially quantitative (94%) native enzymatic activity was regenerated from both SO3-RNase A and its deamidated derivative (SO3-RNase B). Although the two fully active refolded species differ in their elution behavior on ion-exchange chromatography, they are indistinguishable by many other methods. The significance of this finding for studies of the folding of RNase A is discussed.

Amino Acid Sequence↗
PubMed1985

Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide-detection system.

A procedure, developed for the cleavage and reversible blocking of disulfide bonds of proteins by S-sulfonation in preparation for peptide mapping, was applied to ribonuclease A. The complete peptide maps of sulforibonuclease A using limited Staphylococcus aureus protease digestion, tryptic digestion, and tryptic followed by chymotryptic digestion are presented. A description is given of an adaptation of the sulfonation procedure which forms the basis of a sensitive (5-pmol detection limit) and quantitative (+/- 5%) disulfide-detection system for the continuous monitoring of HPLC column effluents for disulfide-containing compounds. The sulfonation procedure, peptide maps, and disulfide-detection system are the key ingredients in a two-dimensional reverse-phase HPLC technique for the determination of disulfide pairings. The applicability of this technique is demonstrated by determining the known disulfide pairings of ribonuclease A. It is also shown that there is no disulfide interchange under the digestion conditions used. This technique is suitable for determining the distributions of disulfide pairings in the intermediates present in the oxidative folding of disulfide-containing proteins.

Animals↗
PubMed1984

Sodium sulfite as an antioxidant in the acid hydrolysis of bovine pancreatic ribonuclease A.

Treatment of hydrochloric acid with sodium sulfite prior to the acid hydrolysis of bovine pancreatic ribonuclease A has been found to suppress the oxidation of cystine, methionine, and tyrosine without adversely affecting the recoveries of other amino acids. Statistical analysis of the results indicated that the assumption of the independence of the mean and the variance, an assumption commonly used in the evaluation of the effects of various treatments, may not be valid in evaluating antioxidants used in the acid hydrolysis of proteins.

Amino Acids↗
PubMed1984

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins.

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.

Chemical Phenomena↗
PubMed1984

Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions.

The C epsilon H proton resonance of His-12 of reduced cysteine S-sulfonated bovine pancreatic ribonuclease A exhibits a nonlinear temperature dependence of the chemical shift in its 1H-NMR spectrum at an apparent pH of 3.0. At temperatures below ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is opposite in sign to those of His-48, His-105, and His-119. At temperatures above ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is similar to those of the other three His C epsilon H resonances. These data indicate the existence of an equilibrium between locally ordered and locally disordered environments of His-12 in the sulfonated protein at temperatures below ca. 35 degrees C. The ordered and disordered conformations interconvert at a rate that is fast relative to the 1H-NMR chemical shift time scale--i.e., the locally ordered structure has a lifetime of much less than 7 msec. These results demonstrate that short- and medium-range interactions can define short-lived local structures under conditions of temperature and solution composition at which the native protein structure is stable. Furthermore, they demonstrate the utility of reduced derivatives of disulfide-containing proteins as model systems for the identification of local structures that may play a role as early-forming chain-folding initiation structures.

Animals↗
PubMed1983

Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

The following peptide was synthesized by classical methods in solution: Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-8). The Michaelis-Menten parameters for the hydrolysis of the Arg-Gly bond in F-8 by thrombin were determined to be Kcat = 31 X 10(-11) M [(NIH unit/L) s]-1 and KM = 310 X 10(-6) M. Comparison of these values with those determined previously for native fibrinogen and for a series of similar synthetic peptides, together with information about the amino acid sequences of this portion of the A alpha chain of abnormal fibrinogens, suggests an important role for Asp at position P10. Differences in the Michaelis-Menten parameters between F-8 and the 51-residue N-terminal CNBr fragment of the A alpha chain of fibrinogen correspond to only 1-2 kcal/mol in binding affinity.

Amino Acid Sequence↗