Acid-hydrolyzed Protein Research Articles

Protein Crosslinking by the Maillard Reaction: Dicarbonyl-Derived Imidazolium Crosslinks in Aging and Diabetes

α-Dicarbonyl compounds that arise from various metabolic pathways react with proteins to form a variety of adducts in a reaction known as the Maillard reaction. These adducts are collectively known as advanced glycation end products or AGEs. Methylglyoxal (MG) and glyoxal (GXL) are two such dicarbonyls. They react with proteins to produce lysine–lysine imidazolium crosslinking AGEs. The imidazolium crosslinks derived from MG (MOLD-methylglyoxal-lysine dimer) and GXL (GOLD-glyoxal-lysine dimer) are present in human tissue proteins. In this study, we report an HPLC method for the simultaneous quantification of GOLD and MOLD in biological specimens. The method consists of reverse-phase HPLC of acid-hydrolyzed proteins, collection of eluate-containing imidazoliums, phenylisothiocyanate derivatization, followed by a second reverse-phase HPLC. This method was linear for both the imidazolium compounds in the range of 0.5–300 pmol. The levels of GOLD and MOLD in aging lenses (20 to 80 years) were trace–8.4 pmol and 15–93 pmol per milligram of protein, respectively. Cataractous lenses showed significantly higher levels of both GOLD and MOLD (mean ± SD, 14.5 ± 1.8 and 141 ± 18.4 pmol per milligram of protein, P < 0.05). Brunescent lenses had the highest levels of imidazolium crosslinks (GOLD, 18.36 ± 2.5; and MOLD, 179.2 ± 32.3 pmol per milligram of protein, P < 0.05). The GOLD and MOLD levels were higher in diabetic plasma proteins when compared to that of normal (GOLD, 17.5 ± 6.34 pmol per milligram of protein vs 43.5 ± 15.96 pmol per milligram of protein; and MOLD, 172.5 ± 32.53 pmol per milligram of protein vs 273 ± 62.67 pmol per milligram of protein, P < 0.05). GOLD and MOLD are significant in terms of tissue damage in aging and diabetes because they represent protein crosslinking by compounds that are major precursors of AGEs. Our method can be used for quantification of imidazolium crosslinks in tissue proteins to assess α-dicarbonyl-mediated protein damage in vivo.

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins.

Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.

Methylation of the polyomavirus major capsid protein VP1

Polyomavirus VP1 has been shown to be modified by phosphorylation, sulfation, acetylation and hydroxylation. The major capsid protein VP1 is now shown to be modified by methylation. Addition of cycloheximide to infected cultures followed by addition of [ 3H-methyl]- l-methionine and subsequent immunoprecipitation, SDS-PAGE and fluorography revealed methylation occurring on VP1. Amino acid analysis of [ 3H]-methyl]- l-methionine-labelled polyomavirus VP1 by two-dimensional paper chromatography and HPLC of the acid-hydrolyzed protein revealed the presence of 3H-labelled trimethyllysine and monomethylarginine.

Hydroxyproline in the major capsid protein VP1 of polyomavirus

Amino acid analysis of [3H]proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of 3H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from [35S]methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

Identification of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine by high-performance liquid chromatography.

Dansyl derivatives of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine were resolved from other amino acids in proteins by the use of high-performance liquid chromatography. The system was tested with amino acid standard combinations as well as with acid-hydrolyzed proteins known to contain methylated residues. In all cases the methylated lysines were well resolved.

Physical and chemical characterization of the major outer membrane protein of Rhodopseudomonas sphaeroides.

The characterization of the major outer membrane protein of Rhodopseudomonas sphaeroides is described. Molecular weight estimations using Ferguson plots derived from sodium dodecyl sulfate and urea-polyacrylamide gels were 39,500 and 32,200, respectively, in good agreement with the value of 33,800 obtained from amino acid compositional studies. NH2-terminal amino acid determinations of the major outer membrane protein revealed a blocked NH2 terminus. Gas chromatography of the acid-hydrolyzed protein confirmed the presence of fatty acid covalently associated with the protein presumably through an amino linkage. Peptide mapping of tryptic and chymotryptic digestions of the protein led to the identification of one peptide in each digest containing fatty acid. Digestion of the fatty acid-containing peptide with pronase resulted in the fatty acid together with L-alanine becoming extractable into hexane. We conclude that the major outer membrane protein of R. sphaeroides is a proteolipid containing at least 1 mol of fatty acid/mol of protein in amide linkage to the NH2-terminal L-alanine of the protein.

A gas-liquid chromatographic-isotope dilution analysis of cysteine, histidine, and tryptophan in acid-hydrolyzed protein

A gas-liquid chromatographic-isotope dilution method for quantitative determination of cysteine (cystine), histidine, and tryptophan and a novel method of protein hydrolysis are described. Acid hydrolysis of protein, using lysozyme as a model, is accomplished in the absence of oxygen and in the presence of 1,2-ethanedithiol for protection of tryptophan. Following the disulfide reduction, cysteine is S-alkylated to protect it during hydrolysis. The resultant tryptophan, alkylated cysteine, and histidine are converted to the methyl esters and then N-acetylated to form volatile derivatives suitable for chromatography on a polar silicone column. Correction for losses is made by an isotope dilution assay. The 70-eV mass spectra of the derivatized amino acids are presented. Ultraviolet spectral data are shown for S-carboxyethylcysteine.

Nuclear accumulation of newly synthesized histones in early Xenopus development

The synthesis of histones, a class of basic nuclear proteins, was examined during embryogenesis in the South African clawed toad. Histones were isolated from nuclear chromatin preparations of pre-gastrula embryos and swimming tadpoles and subjected to acrylamide gel electrophoresis and amino acid analysis. Histone synthesis was demonstrated by the incorporation of 14CO 2 into the amino acid residues of acid-hydrolyzed proteins. Gel electrophoresis indicated that each of the major groups of histones was synthesized in pre-gastrula embryos, as well as in later stages, and incorporated into nuclear chromatin. These newly synthesized embryo histones were similar to those in adult Xenopus laevis liver, when compared by gel electrophoresis and amino acid analysis. Actinomycin D pulse-labeling experiments utilizing the incorporation of [ 3H]lysine into partially dissociated blastulae showed a 20-fold decrease of histone synthesis in actinomycin D treated embryos from that of sibling controls when these basic proteins were separated by Amberlite chromatography. These data suggest that histones are synthesized almost entirely off newly synthesized mRNA blastula (Stages 5–9) in Xenopus laevis.

Promotion of Seed Germination by Cyanide

Potassium cyanide at 3 mum to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form beta-l cyanoalanine, is active in the seeds. beta-l-Cyanoalanine is the most effective of the 23 alpha-amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from beta-cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-(14)C is much lower than of cyanide.Radioactive tracer in K(14)CN shows uptake of about 1.5 mumoles of HCN per gram of A. albus and L. sativa seeds after 20 hours of imbibition. Extracts of the seeds gave high (14)C activity in beta-cyanoalanine, asparagine, and aspartate. The acid-hydrolyzed protein extract gave high activity only in aspartate. Tests were negative for free cyanide in the seed. Respiration of the seed is inhibited more than 75% by KCN and by KN(3) at 10 mm. Azide at greater than 1.0 mm inhibits the promotion of germination by cyanides. Neither 0.1 mm KCN nor KN(3) inhibit O(2) consumption, whereas lower concentrations promote germination. It is concluded that the high rate of utilization of cyanide in the reaction to form beta-l-cyanoalanine and the subsequent incorporation into protein limit any inhibition of oxygen consumption. The promotion of seed germination is substrate-limited by asparagine-aspartate, which is required for protein synthesis.

Conversion of exposed aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues

1. 1. The feasibility of converting exposed carboxyl groups in proteins to carboxamido groups using 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) as a condensing agent was demonstrated. 2. 2. The effectiveness of ammonia as a nucleophile in the EDC-mediated condensation with carboxyl groups in proteins was determined. In 5.5 M NH 4Cl at pH 4.75, 11.5, 14.7, 9.0, 5.0 and 5.1 carboxyl groups are converted to carboxamido groups in chymotrypsinogen A, α-chymotrypsin (EC 3.4.4.5), lysozyme (EC 3.2.1.17), ribonuclease (EC 2.7.7.16) and trypsin (EC 3.4.4.4), respectively. 3. 3. The number of carboxamido groups found was determined from the amount of extra ammonia appearing in acid-hydrolyzed protein which was reacted with EDC and NH 4Cl. In one case, the number of β- and γ-carboxamido groups formed was also determined enzymically by digesting treated protein with proleolytic enzymes and then enzymically assaying the digest for free asparagine and glutamine. Both acid hydrolysis and enzymatic assays gave essentially the same value for the yield of carboxamido groups. 4. 4. 5.5 M NH 4Cl, 1 M glycine methylester, and 1 M glycinamide were all equally effective in modifying carboxyl groups. Since previous studies showed that the glycine derivatives quantitatively modify exposed carboxyl groups in proteins, it was concluded that NH 4Cl may be used to quantitatively convert exposed carboxyl groups in proteins to carboxamido groups. Now that it is possible to convert aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues, it should be possible to establish with greater certainty the role of carboxyl groups in the function and structure of proteins.

Phage formation in Staphylococcus muscae cultures. VIII. Effect of the protein factor and aspartic acid on virus synthesis with various bacterial strains.

1. Four strains of Staphylococcus muscae have been isolated which differ in their growth rates and phage syntheses in Fildes' synthetic medium. 2. Two of the strains when singly infected cannot release phage in Fildes' synthetic medium unless a substance present in certain acid-hydrolyzed proteins is added to the medium. One of these strains also requires other substance(s) present in acid-hydrolyzed proteins in order to grow in Fildes' medium. 3. The two strains which do not require the addition of the phage-stimulating factor have been found either to synthesize this substance, or one similar to it. One of these strains will not grow in Fildes' medium unless substance(s) present in acid-hydrolyzed proteins is added to the medium. 4. The purified acid-hydrolyzed protein factor necessary for virus liberation does not affect the multiplication rate of uninfected S. muscae cells in Fildes' synthetic medium. 5. The substance is not needed for the adsorption or the invasion of the host cell by the virus. In the absence of the factor, the virus is adsorbed to the cell and "kills" it. 6. An analysis carried out by means of the one-step growth curve technique has indicated that the substance is not concerned simply with the mechanism of virus release, but is necessary for some initial stage in virus synthesis. 7. With one bacterial strain not requiring the AHPF, aspartic acid had to be present at least during the minimum latent period for the cell to form virus. 8. In the absence of aspartic acid, the virus was adsorbed to the cell and killed it, but no virus was released from singly infected bacteria. 9. If the cells were grown in a medium containing aspartic acid and then resuspended in the medium minus aspartic acid, no virus was released, although such cells contained at least two times the amount of aspartic acid necessary for the burst size in the complete medium. 10. Aspartic acid, a constituent of the virus particle, appears from an analysis of one-step growth curves to take part in the initial phase of phage synthesis. 11. The effect of amino acids on virus formation is discussed in relation to the time sequence of virus protein and desoxyribonucleic acid synthesis.

Phage formation in Staphylococcus muscae cultures. IX. Effect of multiple infection on virus synthesis in the absence and presence of specific substrates.

1. A strain of S. muscae which requires a substance present in certain acid-hydrolyzed proteins (AHPF) for virus liberation when singly infected in Fildes' synthetic medium no longer needs this substance when multiply infected. 2. In the absence of the AHPF under conditions of multiple infection the amount of phage released is approximately equal to the number of infecting particles between two to ten. Over ten particles per cell has no further effect on the yield of virus. 3. The experimental evidence indicates that it is the phage particle and not some other component in the lysate which can replace the AHPF. 4. The minimum latent period and rise period of cells singly infected in the presence of the AHPF and multiply infected in the absence of the AHPF are the same. 5. The desoxynucleic acid synthesis of cells, infected with a very few virus particles in the presence of excess AHPF and multiply infected with ten particles in the absence of the AHPF, occurs at approximately the same rate, with both infected samples synthesizing about the same amount of desoxynucleic acid and liberating the same yields of virus. 6. A strain of S. muscae which requires aspartic acid for virus synthesis when singly infected does not need this substance when multiply infected, the burst size under the latter conditions depending upon the multiplicity of infection between 3 to 12 particles per cell. 7. The data indicate that the virus released from multiply infected cells in the absence of added AHPF or aspartic acid is newly synthesized virus and not the original infecting particles. 8. The phage particle contains the AHPF and aspartic acid. 9. As a tentative working hypothesis, it is assumed that the AHPF and aspartic acid for phage formation under conditions of multiple infection, in the absence of added AHPF, or of aspartic acid, are contributed by the original infecting particles. 10. Ultraviolet-inactivated phage is adsorbed to the host cell and kills the cell although little virus is released under the experimental conditions. 11. Ultraviolet-inactivated phage particles, if added before the active particle is adsorbed, will greatly inhibit the liberation of new virus particles; but does not do so if added a few minutes after the active particle has been adsorbed. 12. Under the experimental conditions, reactivation of phage when present in multiply infected cells does not occur; and such ultraviolet-inactivated phage cannot serve as a source of the AHPF or aspartic acid, although the AHPF can be liberated from such inactivated particles by acid hydrolysis. 13. The results are discussed in relation to Luria's experiments with ultraviolet-treated phage and to his "gene pool" hypothesis of phage formation.