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Describes an application of two-dimensional, nuclear magnetic resonance techniques for the study of proteins. Amino acid residue; Molecular weight; Glycyl residue spin systems; Spin-spin coupling constants; Protein conformation.

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

The transformation of aerosol proteinaceous matter (PrM) has potentially adverse environmental and health effects. The origins and atmospheric processes of PrM have obtained an increasing interest, but there are still large uncertainties. Although aerosol liquid water (ALW) has been found to be vital in numerous atmospheric processes, its impact on atmospheric degradation of PrM remains poorly understood. ALW, proteins, and low molecular weight PrM (LMW PrM, <10 kDa) were investigated in fine aerosols (PM2.5) collected in urban and suburban Nanchang (Eastern China) over a 1‐year period. The average concentrations of proteins and LMW PrM in the urban center were higher than those in the suburban area. Urban PrM tended to increase from cool season to warm season, which was opposite to the case of suburban PrM. These differences could be attributed to varied sources and atmospheric processes of PrM. Increase in ALW was prominent from the suburban site to the urban site, which was attributable to increased anthropogenic nitrate and sulfate. LMW PrM can be degraded from higher molecular weight PrM by ozone. Furthermore, correlation analysis between LMW PrM and ozone and ALW suggested that the enhanced ALW in the urban center facilitated the ozone‐induced LMW PrM release. The ALW‐related protein degradation was further supported by ozone exposure experiments with bovine serum albumin and PM2.5 samples under dry condition. Our findings suggest that ALW is a crucial promoter during PrM degradation by ozone, providing new insights into atmospheric transformation of proteins. Plain Language Summary: Recent observation‐based studies on interactions between aerosol proteins and ozone have suggested that free amino acids in fine aerosols (PM2.5) can be degraded from proteins under the influence of ozone. Here, proteins, low molecular weight (LMW) proteinaceous matter (PrM), and aerosol liquid water (ALW) were investigated in PM2.5 collected in urban and suburban Nanchang (Eastern China) over a 1‐year period. Interestingly, we found that alone ozone cannot explain the production of LMW PrM in this study. Specifically, LMW PrM was found to be largely degraded from higher molecular weight PrM by ozone in the urban center (high ALW), whereas this degradation process was insignificant in the suburban area (low ALW). Correlation analysis between LMW PrM and ozone and ALW suggested that increased ALW in the urban center may promote the production of LMW PrM from ozone‐related aqueous‐phase reactions. This consideration was also supported by ozone exposure experiments with bovine serum albumin and PM2.5 samples under dry condition. The overall results suggest that ALW plays an important role in PrM degradation by ozone. Therefore, this study provides new insights into the fate of proteins in the atmosphere. Key Points: The significant increase in aerosol liquid water (ALW) from the urban center to the suburban area is attributable to increased anthropogenic nitrates and sulfatesAtmospheric degradation of high molecular weight proteinaceous matter (PrM) by ozone is an important secondary source of low molecular weight (LMW) PrM, especially in the urban areaThe increased ALW in the urban area promotes the ozone‐related LMW PrM release [ABSTRACT FROM AUTHOR]

Two 1,3; 1,4-β-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3; 1,4-β-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants. [ABSTRACT FROM AUTHOR]

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I is composed of six subunits of a molecular weight 60,000. Furthermore, quantitative determination of the N-terminal amino acids indicates the presence of six methionine residues per mole of native enzyme (mol wt, 360,000). Equilibrium sedimentation studies of N-ethylmaleimide treated protein shows that its six disulfide bridges, revealed by chemical analysis, are intra-chain. Fingerprints of tryptic digests of the protein fail to reveal any difference between the subunits. [ABSTRACT FROM AUTHOR]

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I has been obtained in a homogeneous state from extracts of Eseherichia coli K 12. The molecular weight of the protein has been determined by sedimentation equilibrium to be 360,000 dalton. The amino acid composition is given. Titration of sulfhydryl groups by 5-5'-dithiobis- (2-nitrobenzoic)acid leads to an estimation of 16 to 18 reactive cysteines per mole of the protein, all of which can be protected by addition of the allosteric inhibitor, L-threonine. [ABSTRACT FROM AUTHOR]

Summary: The aim of this study was to develop a green ultrasound‐assisted enzymatic process to separate protein from brewer's spent grain (BSG) to produce protein hydrolysates and determine the physicochemical properties of produced protein hydrolysates. When the enzyme (Alcalase) loading increased from 1 to 20 μL g−1 BSG, the protein separation efficiency increased from 34.0% to 61.6%. The application of ultrasound pretreatment further increased protein separation efficiency to 69.8%. More promisingly, the ultrasound pretreatment was able to reduce enzyme loading by 73% and decrease enzyme incubation time by 56%. The produced protein hydrolysates had molecular weights lower than 15 kDa and high protein solubilities at the pH of 1.0–11.0. The ultrasound pretreatment improved the protein solubility to above 90%. Glutamic acid and proline were the most abundant amino acids in produced protein hydrolysates. This study demonstrated that enzymatic hydrolysis along with ultrasound pretreatment is an effective way to separate protein from BSG. [ABSTRACT FROM AUTHOR]

Autographa californica nucleopolyhedrovirus orf74 (Ac74) is located between 62 311 and 63 108bp in the AcMNPV genome, which encodes 265 amino acid residues with a predicted 31 kDa molecular weight. The homologues of Ac74 were searched using BLASTP in protein databases, GenBank/EMBL and SWISS-PROT. The result revealed that deduced Ac74 protein was homologous to the predicted products from 10 lepidoptera NPV ORFs. The multiple sequence alignments of Ac74 and its 10 homologues manifested only one amino acid residue was completely conserved. The transcript analysis revealed that the transcript of Ac74 was detected from 24–72 hours post-infection (hpi). The product of Ac74 was detected at 24 hpi and lasted until 72 hpi by Western blot using anti-Ac74 antiserum, consistent with reverse transcriptase polymerase chain reaction results. These results suggested Ac74 was expressed during the later stages of infection. The product of Ac74 was 31 kDa in size, consistent with predicted molecular weight. The subcellular localization of Ac74 proteins manifested Ac74 protein in the cytoplasm, and was hardly present in the nucleus at 24 hpi. The fluorescence was also observed in polyhedra, except cytoplasm at 72 hpi. Together, Ac74 is a functional protein with 3 1kDa molecular weight and is located in the cytoplasm and the polyhedra. [ABSTRACT FROM AUTHOR]

The major polypeptide chain of bovine β-crystallin, βBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the βBp chain. The N-terminal alanine residue was shown to be N-α-acetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23 210. There is a considerable degree of homology between the N-terminal and C-terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of βBp is sufficiently related to that of γ-crystallin II to place these proteins in the same superfamily. No sequence relationship was found with the α-crystallin chains. [ABSTRACT FROM AUTHOR]

Using 14C-labeled Salmonella bacterial cells as the substrate, the enzymic and molecular properties of the base-plate parts of phage P22 were studied. The base-plate part consisted of a single protein species which cleaved extensively the O-antigen of Salmonella typhimurium, Salmonelly schottmuellerie and with somewhat slower rate that of Salmonella typhi, releasing oligosaccharide products with rhamnose at the reducing end. Much less cleavage was observed with a strain of S. typhimurium lysogenie for P22, and no significant reaction with Salmonella anatum. Salmonella newington and Salmonella minneapolis. The base-plate part enzyme was a very heat-stable protein and only 10–20% loss was observed after treatment at 85 °C for 5 min. The pH optimum of the enzyme was around 7.5. and the glycosidase activity was not influenced by the ionic strength (25–250 mM) of the medium or the presence of Mg2+. The molecular weight of the base-plate part was 320000 by sedimentation equilibrium. Dodecylsulphate-acrylamide gel electrophoresis revealed a single band of molecular weight 77000, indicating that a single base-plate part corresponds to a tetramer of identical subunits. Circular dichroism spectra of P22 base-plate parts showed a major contribution of β structure. The protein was rich in acidic amino acids, glycine and serine. [ABSTRACT FROM AUTHOR]

Histidine decarboxylase from Micrococcus sp.n. was carboxymethylated and two types of subunits, I and II, were separated in excellent yield by exclusion chromatography on Sephadex G-100 in 8 M urea. Total compositions, number of tryptic peptides and N-terminal groups of the subunits were analyzed. The larger subunit I is composed of all amino acids commonly found in proteins and has a blocked N-terminal residue, whereas the smaller subunit II does not contain proline, histidine or cysteine and has N-terminal methionine. The number of tryptic peptides detected in peptide mapping experiments of subunit I is about 27 and that of subunit II is about 15. These figures are in good agreement with the total number of lysine and arginine residues determined by amino acid analysis, if subunit I contains about 270 residues, corresponding to a molecular weight of 29000, and subunit II about 70 residues, corresponding to a molecular weight of 7000. It is concluded that histidine decarboxylase contains two apparently homogeneous but completely different polypeptide chains and that the native enzyme of molecular weight 110000 is a hexamer with three subunits of each type. [ABSTRACT FROM AUTHOR]

In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 2% aqueous acetic acid. The exact was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G-25. The high-molecular-weight fraction was chromatographed on SE-Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained. The mobile was homogeneous by standard criteria of poem purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is 5000. Phoratoxin has the following amino acid composition (residues/mole): alanine 2, arginine 3, aspartic acid 4, &frac12-cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine 1, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine 1. The molecular weight, calculated from the amino acid composition, is 4884. The material contains no reducing sugars, amino sugars or sialic acids. The LD50, determined by intraperitoneal injection m mice, is 0.57 ± 0.05 mg/kg bodyweight. [ABSTRACT FROM AUTHOR]