Haptoglobin-hemoglobin complex. Subunit interaction probed by cross-linking.

J Greer; W D Liao; W E Brown

Home/Archive/PubMed

PubMed · 7263686

Haptoglobin-hemoglobin complex. Subunit interaction probed by cross-linking.

J Greer·W D Liao·W E Brown

View original source ↗Export metadata ↓

Abstract

Haptoglobin-hemoglobin complex has been cross-linked using the bifunctional reagent 1,5-difluoro-2,4-dinitrobenzene. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that specific cross-linking was obtained between haptoglobin H chain and a hemoglobin chain. The cross-linked complex was reduced and denatured, and the cross-linked subunits were separated from the unreacted haptoglobin and hemoglobin chains by molecular sieve chromatography. Peptide analysis on the purified cross-linked subunits showed that haptoglobin H chain was cross-linked to hemoglobin beta chain only. This result indicates that the H and beta chains are in close proximity in the haptoglobin-hemoglobin complex.

Explore related subjects

Amino Acids Cross-Linking Reagents Dinitrofluorobenzene Electrophoresis, Polyacrylamide Gel Haptoglobins Hemoglobins Humans Macromolecular Substances Peptide Fragments Trypsin Journal Article Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

J Greer, W D Liao, W E Brown. 1981-08-25. Haptoglobin-hemoglobin complex. Subunit interaction probed by cross-linking.. https://pubmed.ncbi.nlm.nih.gov/7263686/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Chimeric structural isomer fragments as cost-efficient internal standards for amino acid quantification by mass spectrometry.

Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.

Amino Acids↗

PubMed1995

Comparison between the phi distribution of the amino acids in the protein database and NMR data indicates that amino acids have various phi propensities in the random coil conformation.

It has been indicated that amino acids have various intrinsic phi and psi propensities, as demonstrated from the comparison between experimental secondary structure propensities and their relative statistical distribution in the protein database for the appropriate region of the Ramachandran plot. However, this does not eliminate the possibility that these experimental propensities are the result of context effects due to the secondary structure environment of the mutated position. To demonstrate that there are at least real intrinsic phi propensities, independent of context effects, we have used two different nuclear magnetic resonance parameters related to the phi dihedral angle (J3 alpha HN coupling constants and the chemical shift of the C alpha H proton), determined in random-coil tetra- and pentapeptides, and/or in proteins. Comparison of the experimentally determined values for these parameters with the theoretical ones determined from the analysis by different empirical and theoretical equations of the phi dihedral angle statistical distribution of the amino acids in the protein database, supports the idea that each amino acid has, at least, different phi intrinsic propensities. Consideration of all conformations, or only coil conformations, in the protein database produces similar results. The reasonable correlation between these experimental and theoretical data and the hydrogen-exchange data in random-coil peptides suggests that maximisation of hydrophobic surface-buried and hydrogen-bond formation with the solvent could be responsible for these different random-coil conformational preferences. Analysis of the intrinsic propensities for beta-strand, alpha-helix and polyproline II dihedral angles of the 20 amino acids in coil conformations, indicates that the side-chain of the amino acids is mainly determining the relative preferences for the phi angle.

Amino Acids↗

PubMed1995

A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.

A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗