John Hempel

By-products as an aid in residue identification during peptide sequence analysis with dimethylaminoazobenzene isothiocyanate

Abstract4-N,N-Dimethylaminoazobenzene 4′-isothiocyanate degradations permit sensitive and fast manual sequence analysis, but assignments of some residues are difficult. Hydrophobic residues, especially leucine and isoleucine, are badly resolved on polyamide thin-layer chromatography. Differently colored by-products have been described before for a few labile residues, but it is now shown that most residues can give rise to characteristic by-products. These have different colors and chromatographic properties, depending on the nature of the parent residue. Thus, two to three sets of spots characterize each residue, giving multiple identification with increased reliability. Although variable and dependent on chemicals and conditions, by-products are often prominent after conversion with 50% trifluoroacetic acid, and can be utilized to improve the identifications.

Structures of human alcohol and aldehyde dehydrogenases.

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.

Human liver mitochondrial aldehyde dehydrogenase: a C-terminal segment positions and defines the structure corresponding to the one reported to differ in the Oriental enzyme variant

Mitochondrial isoenzyme Amino acid sequence Isoenzyme difference Structure‐function relationship

Atypical human liver alcohol dehydrogenase: the β 2-Bern subunit has an amino acid exchange that is identical to the one in the β 2-Oriental chain

Alcohol dehydrogenase Human liver isoenzyme Primary structure Mutation

Internal chain cleavage and product heterogeneity during Edman degradation of isosteric peptide analogs lacking the α-carbonyl function

A synthetic peptide analog, with one peptide carbonyl group replaced by a methylene bridge, was submitted to structural analysis by Edman degradation. Multiple cleavages were obtained in the first cycle, due to phenylthiocarbamylation of the internal secondary amine as well as spontaneous alkaline cyclization and subsequent recoupling with the Edman reagent. Three fragments from cleavage of the peptide analog after a single Edman cycle were purified by reverse‐phase high‐performance liquid chromatography. The results support previous observations in a novel combination. The reactions may also be important with native polypeptides since non‐quantitative alkaline cyclization now encountered can mimic apparent N‐terminal heterogeneity in agreement with earlier data, while quantitative cyclization can mimic loss of N‐terminal residues.

Structural characteristics of a major seed albumin ofPisum sativum

The major pea seed albumin fromPisum sativurn was carboxymethylated, cleaved with CNBr, and submitted to sequence analysis of the fragments in order to characterize the structural organization of the protein chains. Four major pools of largely homogeneous CNBr fragments were obtainede and likely N-and C-terminal fragments were identified. StruCtural analysis suggested the presence of single positions with microheterogeneities. It also revealed structures with long segments of distinct homology (52% structural identity), indicating the presence of different but related protein chains, or less likely, of repetitive structural elements within a chain. However, preparations appear largely homogeneous in protein class, and contain similar polypeptide chains of about 200 residues in mainly hydrophilic structures, with few methionine and cysteine/half-cystine residues.

Cleavage at acyl-proline bonds with sodium in liquid ammonia: Application with nanomolar amounts of peptides and separation of products byhigh-performance liquid chromatography for structural analysis

Cleavage of X-Pro bonds with metallic sodium in liquid ammonia is a little-used method due to difficulties with handling of reagents, variable cleavage yields, and separation of peptides from salt byproducts. Construction of a small distillation/reaction apparatus permitted peptide incubations at the nanomole scale. Reverse-phase high-performance liquid chromatography (HPLC) of the residue after removal of NH3 allows separation of the salts and fractionation of the cleaved peptides which may be taken directly for sequence analysis. HPLC also allows rapid assessment of the degree of cleavage before structural analysis. Cleavage of some peptides proceeded in high yield while others were cleaved poorly or not at all, modifying earlier generalizations on factors influencing cleavage.