Biomedical subjects

B T Chait

Publications and source records attributed to B T Chait.

At least 127 records · Page 7Linked to original sources

PubMed1991

Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.

Allosteric Regulation↗

PubMed1991

Use of a single-quadrupole mass spectrometer for collision-induced dissociation studies of multiply charged peptide ions produced by electrospray ionization.

The feasibility of obtaining the collision-induced dissociation (CID) spectra of multiply charged peptide ions produced by electrospray ionization in a simple and inexpensive single-quadrupole mass spectrometer is demonstrated. Collisional activation was carried out in the high-pressure region between the capillary exit and the skimmer entrance to the mass analyzer. The CID of multiply charged peptide ions is very efficient, and the observed fragment ion intensities are typically 1-5% of the parent ion intensity prior to CID. About 70 pmol of the peptide is consumed in obtaining each CID spectrum. Spectra obtained by CID of multiply charged ions from bradykinin, angiotensin II, two peptides with features similar to tryptic peptides, and a synthetic analogue of a component of TGF-alpha containing two disulfide bonds are shown. The influence of the primary structure of the peptide on the observed fragmentation pathways is discussed. Although the present single-quadrupole configuration is simple and effective, the inability to choose a particular parent ion for collisional activation makes it less powerful than the triple-quadrupole configuration for mixtures of peptides and peptide samples that yield more than one charge state in the normal mass spectrum. However, it has the potential for inexpensively obtaining sequence information of proteins at high sensitivity by analyzing the pure tryptic peptides obtained by on-line or off-line chromatographic separation of tryptic digests.

Amino Acid Sequence↗

PubMed1991

Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.

Hydrogen-exchange electrospray-ionization mass spectrometry is demonstrated to be an effective new method for probing conformational changes of proteins in solutions. The method is based on the mass spectrometric measurement of the extent of hydrogen/deuterium exchange that occurs in different protein conformers over defined periods of time. Results are presented in which hydrogen-exchange electrospray-ionization mass spectrometry is used to probe conformational changes in bovine ubiquitin induced by the addition of methanol to aqueous acidic solutions of the protein.

Animals↗

PubMed1990

Analysis of mixtures of closely related forms of bovine trypsin by electrospray ionization mass spectrometry: use of charge state distributions to resolve ions of the different forms.

Electrospray ionization mass spectrometry has been utilized for the analysis of bovine trypsin. Commercial active bovine trypsin is comprised of a mixture of three closely related different forms: beta-, alpha-, and psi-trypsin with similar molecular masses (23293, 23311, and 23329 mass units respectively). Peak broadening caused by the natural isotopic abundance of atoms in the protein molecules results in overlap of the ion peaks from the different trypsin forms. Therefore, accurate determination of the molecular masses of these trypsins in a mixture is not straightforward. However, because the trypsin isoforms have different number of basic groups and different higher order structures, the different forms acquire different numbers of charges. Therefore, ions from the different trypsins appear in different parts of the mass spectrum allowing accurate determination of their molecular masses.

Animals↗

PubMed1990

High-accuracy molecular mass determination of proteins using matrix-assisted laser desorption mass spectrometry.

A method for obtaining protein molecular masses with an accuracy of approximately +/- 0.01% by matrix-assisted laser desorption using an internal calibrant is described. The technique allows accurate mass determinations of protein sample sizes as small as 1 pmol. High concentrations of organic and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal intensity or the mass assignment. The ability to assign an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule ions in the mass spectrum.

Animals↗

PubMed1990

Retinol is essential for growth of activated human B cells.

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.

B-Lymphocytes↗

PubMed1990

Electrospray ionization mass spectrometric peptide mapping: a rapid, sensitive technique for protein structure analysis.

Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure.

Apolipoprotein A-I↗

PubMed1990

An electrospray-ionization mass spectrometer with new features.

The construction and performance of an electrospray-ionization mass spectrometer with new features are described. The mass spectrometer consists of a newly designed electrospray ion-source that is plugged directly into a modified commercial quadrupole mass spectrometer with the ions entering the mass analyzer through a long metal capillary tube and three stages of differential pumping. The present ion source differs from previous designs in the combination of techniques employed in the transportation and desolvation of solvated biomolecule ions, prior to mass analysis. Transport of ionized entities between atmospheric pressure and vacuum is carried out through a 203 mm long stainless steel capillary tube with a 0.5 mm bore. Desolvation is effected by the use of controlled heat transfer through the long capillary tube and collisional activation in a region of reduced pressure between the capillary tube exit and the skimmer. Desolvation with this system is convenient and effective and does not involve the strong countercurrent flows of gases that have been used by all previous workers. The effects on the spectra of peptides of capillary tube temperature and desolvation collision energy are investigated. Electrospray-ionization mass spectrometric results are described for thirteen proteins with molecular masses ranging from 5000 to 77,000 Da. The performance of the present instrument, with respect to mass accuracy and sensitivity, is comparable with previously reported systems. The effect of protein concentration in solution on the electrospray mass spectrometric response and charge-state distribution is discussed.

Mass Spectrometry↗

PubMed1990

Rapid, sensitive analysis of protein mixtures by mass spectrometry.

We have developed a method for determining the molecular masses of proteins in complex mixtures by mass spectrometry. The method has the capacity to examine the components of mixtures without using any chromatographic separation steps and will tolerate relatively large amounts of buffers and inorganic contaminants. It allows the simultaneous determination of protein molecular masses from 1 to 40 kDa with an accuracy of +/- 0.01% and above 40 kDa with reduced accuracy. The lower limit for practical detection of a protein is a concentration of approximately 0.1 microM, and less than 1 microliter of such a solution is consumed. The analysis is very fast: less than 15 min is necessary to perform the complete analysis, including sample preparation, introduction into the mass spectrometer, mass spectrum collection, and data reduction. The mass spectrum that is obtained does not require elaborate interpretation because there is no fragmentation of the ionized protein (or protein subunit) molecule. Therefore, there is a one-to-one correspondence between the peaks in the mass spectrum and the proteins present in the original mixture. The spectra assume the appearance of chromatograms, with the abscissa being mass-to-charge ratio rather than chromatographic retention time.

Animals↗

PubMed1990

High resolution high performance liquid chromatography fingerprinting of purified human chorionic gonadotropin demonstrates that oxidation is a cause of hormone heterogeneity.

To characterize the structures of the various forms of hCG and its immunologically related fragments measured in blood, urine, and tissue culture media, we developed a highly sensitive HPLC tryptic fingerprinting system that can characterize as little as 15 micrograms of material. Standard preparations of the subunits of hCG purified from pregnancy urine were found to exhibit heterogeneity on reverse phase HPLC chromatography, complicating efforts to characterize such forms of hCG. The same type of chromatographic heterogeneity was observed when the hormone was reduced and S-carboxymethylated (RCM) and its RCM subunits separated on HPLC. One major and one minor peak were observed in the alpha- and beta-subunits from both native and RCM hormone, with a ratio of between 5:1 and 10:1. Development and application of a high resolution, semimicrobore HPLC fingerprint technique used together with an examination of selected peptides by amino acid sequencing and mass spectrometry defined the differences between these forms as oxidation. Using each of the isolated RCM subunit's major and minor HPLC peaks, the oxidation was shown to be reversible and probably due to interconversion of methionine with its sulfoxide form.

Chorionic Gonadotropin↗

PubMed1990

Structural studies of a hornet venom allergen antigen 5, Dol m V and its sequence similarity with other proteins.

The primary structures and the disulfide-bond pairings of two chromatographic forms A and B of a venom protein from white-faced hornet (Dolichovespula maculata) were determined. This protein, designated as antigen 5 (Dol m V), has 204-205 amino acid residues and it contains 4 disulfide bonds. This protein is an allergen in man but its biologic function in hornet is not yet known. Dol m V is found to have partial sequence similarity with pathogenesis-related proteins from tobacco and tomato leaves and with a sperm-coating protein from rat epididymis.

Amino Acid Sequence↗

PubMed1989

Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. Thermodynamic and functional consequences.

Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.

Amino Acid Sequence↗

PubMed1989

A mass spectrometric technique for detecting and identifying by-products in the synthesis of peptides.

The utility of a new mass spectrometric technique for detecting and identifying peptide by-products produced in the synthesis of peptides is demonstrated. The technique involves three sequential steps: (1) practically nondestructive 252Cf plasma desorption mass spectrometric analysis of monolayer amounts of the peptide(s) of interest bound to a thin layer of nitrocellulose; (2) enzyme-catalyzed microscale chemical reaction of the surface-bound peptide(s) to produce structurally informative hydrolysis products; (3) plasma desorption mass spectrometric analysis of these hydrolysis products. The first step determines the presence and the molecular weights of unwanted by-products resulting from errors or incomplete reactions during synthesis. The subsequent two steps provide information on the precise location in the peptides where errors have occurred. In the present paper, the technique is applied to an investigation of unwanted peptide by-products associated with the use of tryptophan during stepwise solid-phase peptide synthesis. Synthetic preparations of melittin and [Bpa-8]dynorphin A (1-17) were each found to contain a major impurity with molecular weight 28 Da higher than that of the desired product. The impurity in the melittin preparation, in which the final deprotection step involved the high-low HF procedure, was shown to result from incomplete removal of the formyl group from Trp-19. On the other hand, the impurity in the [Bpa-8]dynorphin A (1-17) preparation, where the removal of the formyl group from Trp-14 was carried out using piperidine, was shown to result from migration of the formyl group to Lys-11 or Lys-13.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗

PubMed1989

Factors affecting the ultraviolet laser desorption of proteins.

The production of high-mass quasimolecular ions from proteins by matrix-assisted ultraviolet laser desorption is described. A simple time-of-flight system using a Q-switched frequency-quadrupled Nd-YAG laser to desorb protein molecules is shown to have a mass range of up to 116,000 u by the observation of intact, singly charged quasimolecular ions from 700 fmol of beta-galactosidase subunit (mol.wt = 116,336 Da). Both positive- and negative-ion spectra of proteins are shown. Four new matrix materials, with properties as good as or better than nicotinic acid, are described. A mass resolution of approximately 500 (full width at half maximum definition) is demonstrated for proteins with mol.wt less than 20,000 Da. Product species, formed by fast photochemical reactions in the matrix, are observed to form adduct ions with protein molecules. These adduct ions are a significant cause of the observed broadness of protein quasimolecular ion peaks. The practical physical considerations in detection of large-mass quasimolecular ions from laser desorption, such as detector overloading, are discussed.

Animals↗

PubMed1989

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins.

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).

Animals↗

PubMed1989

Matrix-assisted laser-desorption mass spectrometry using 355 nm radiation.

Matrix-assisted laser desorption of proteins with the 355 nm frequency-tripled output of a neodymium:yttrium-aluminium-garnet laser has been demonstrated. In order to produce desorption at this wavelength, the cinnamic acid derivatives ferulic, caffeic and sinapinic acids have been employed as matrices. Excellent sensitivity (routinely one pmol of protein) and good mass resolution (m/delta m 400, Full width at half maximum) have been obtained by this new method. Ultraviolet photo-spectrometry of the cinnamic acid derivative matrices suggests strongly that they can be used as protein laser-desorption matrices at any wavelength between 260 nm and 360 nm, allowing the development of laser-desorption mass spectrometers based on nitrogen or excimer lasers.

Caffeic Acids↗

PubMed1989

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha.

Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.

Amino Acid Sequence↗

PubMed1989

Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein. Resolution of cyclized peptides by HPLC and characterization by mass spectrometry.

RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP-PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino-terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino-terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP-PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37 degrees, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0 degree, in all the three buffers. Thus, an extended digestion at 0 degree or a brief digestion at 37 degrees in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino-terminal glutamine residues of PepM49 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗