Paul M. Gallop

DETERMINATION OF CARBONYL COMPOUNDS WITH N-METHYL BENZOTHIAZOLONE HYDRAZONE.

Abstract N -Methyl benzothiazolone hydrazone reacts with various carbonyl compounds. The azine and “osazine” derivatives formed under defined conditions have characteristic spectra which permit the identification of saturated and unsaturated aldehydes and ketones, keto acids, and many other related compounds in a simple spectrophotometric assay. The azines of aldehydes can be further reacted with the oxidized form of MBTH to give rise to tetraazopentamethine cyanine dyes used in a colorimetric procedure. The sensitivity of both methods is such that microgram amounts of a carbonyl compound can be identified and measured rapidly and accurately. Since carbohydrates having pyranose structures are unreactive in the colorimetric and spectrophotometric methods, many aldehydes and ketones can be measured in their presence.

The nature of crosslinking in collagens from mineralized tissues

Abstract Following reduction with sodium borotritide, bone and dentine collagens are found to contain only a few labelled substances, in contrast with the soft tissue collagens. The reduced aldehydes, ϵ-hydroxynorleucine (HNL) and σ, ϵ-dihydroxynorleucine (DHNL) are abundant in the mineralized collagens and their relative proportions differ in fetal and mature tissues. The reduced crosslinks, σ-hydroxylysinonor leucine (HLNL) and σ, σ-dihydroxylysinonorleucine (DHLNL) together account for a major portion of the radioactivity and their relative abundance varies with age. In contrast with the results of others, we find that DHLNL is the major reducible crosslink of mineralized collagens, comprising up to 60% of the total radioactivity. Moreover, reduction with NaBD4 shows that a portion of the Schiff base precursor of DHLNL becomes reduced in vivo. The marked insolubility of bone and dentine collagens may, in part, be attributed to such in vivo reduction.

Modification and introduction of a specific radioactive label into the erythrocyte membrane sialoglycoproteins

Abstract A specific radioactive label was introduced into the sialoglycoproteins of the erythrocyte membrane by sequential sodium periodate oxidation and tritiated sodium borohydride reduction. This was achieved whether the sialoglycoproteins were isolated, present in situ within the intact erythrocyte, or the isolated erythrocyte membranes. The label is found in the oligosaccharide chains of the sialoglycoproteins predominantly in residues which were formerly those of bound sialic acid. The method appears selective for the sialoglycoproteins and certain, as yet unidentified lipid components.

Isolation of Hydroxylysinonorleucine and its Lactone from Reconstituted Collagen Fibrils

Summary Chromatographic fractionation of acid hydrolysates of N aBH4-reduced reconstituded collagen fibrils was carried out. Two compounds were isolated and shown to be hydroxylysinonorleucine and its lactone. The lactone underwent dehydration under certain circumstances yielding two new compounds which by mass spectrometry were both found to be Δ3,4-dehydrolysinonorleucine. The two compounds are probably the cis, trans isomers of Δ3,4-dehydrolysinonorleucine.

Aldol-histidine, a new trifunctional collagen crosslink

Abstract A new trifunctional crosslink, 2,10-diamino-5-hydroxymethyl-6-(N-1-histidyl)-undecandioic acid, termed aldol-histidine, was isolated from borohydride reduced cow skin insoluble collagen. The compound was characterized by pmr spectroscopy and mass spectrometry of several volatile derivatives. The crosslink may be derived from Michael addition of a histidine residue to the known aldol crosslink, 2,10-diamino-5-formyl-5-undecandioic acid. This is the first example of a histidine containing crosslink found in structural protein.

Isolation of a glycoprotein--glycolipid fraction from human erythrocyte membranes.

Summary An ethanol fractionation procedure was developed for the isolation of glycoproteins from aqueous pyridine solubilized human erythrocyte membranes. The preparation resembles that obtained by extraction with phenol in its amino acid and carbohydrate composition and the presence of antigens. In addition to the glycoproteins the preparation contains glycolipid components.

The presence in elastin of possible cyclic precursors of desmosine and isodesmosine.

Abstract Mass spectral evidence is presented that the elastin of bovine ligamentum nuchae contains, in addition to desmosine and isodesmosine, larger levels of their respective cyclic dehydrodesmopiperidine precursors. In addition to being precursors of the desmosines, such compounds are most likely cross-links in themselves. In fact, the occurrence of these compounds, now isolated as their reduced derivatives, may clarify the discrepancies in the stoichiometry of the conversion of lysine residues to cross-linking components as noted by several workers (1,2,3). With this study the occurrence of all of the tetrafunctional amino acid cyclic structures related to the desmosines is now confimed by mass spectrometry.

Reductive desulfuration and mass spectrometric sequencing of sulfur-containing peptides

Abstract The difficulties presented by sulfur-containing amino acids in mass spectrometric sequencing of peptides can be eliminated by reductive desulfuration in the presence of an easily prepared and efficient Ni2B catalyst. The procedure described consisting of reductive desulfuration, isobutoxycarbonylation and permethylation allows simple and rapid modification of small levels of peptides, and advances the potential of mass spectrometry as a tool in peptide sequencing procedures.

Dehydromerodesmosine and merodesmosine in elastin

Summary Elastin of bovine ligamentum nuchae was prepared without the use of hot alkali and subsequently reduced either with 3[H]NaBH4 or 3[H]NaBD4. Reduction with NaBD4 permitted the measurement of the ratio of merodesmosine to dehydromerodesmosine in natural elastin, and in this case was found to be approximately one. Thus in ligamentum elastin, untreated with hot alkali and/or borohydride, two kinds of Schiff base crosslinks, dehydrolysinonorleucine and dehydromerodesmosine, have been shown to occur as such. They also occur naturally in the respective forms of their reduced derivatives, lysinonorleucine and merodesmosine.

Sequence analysis of arginyl peptides by mass spectrometry

Abstract The problem of modifying arginine-containing peptides to form volatile derivatives suitable for mass spectrometry has recently been solved in two general ways. Treatment of arginine-containing peptides with hydrazine yields ornithine residues (1, 2) while treatment with various dicarbonyl reagents results in the formation of a heterocyclic ring (1, 3). In this communication we report the use of 1,2-cyclohexanedione (CHD) in modifying arginine residues for mass spectrometry. This reagent has been shown to react quantitatively with arginine residues of proteins (Fig. 1) without significant side reactions or peptide bond cleavage (4). By combining this modification of arginine residues with acylation and permethylation (2, 5, 6) volatile compounds were obtained from several peptides. The mass spectra of these compounds combined with knowledge of the amino acid composition of the parent peptide yielded considerable sequence information for each of the arginine-containing peptides studied, including the nonapeptide bradykinin.