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1.
An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin 总被引:4,自引:0,他引:4
Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage. 相似文献
2.
The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated. 相似文献
3.
Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.
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K Yanai N Takaya N Kojima H Horiuchi A Ohta M Takagi 《Journal of bacteriology》1992,174(22):7398-7406
Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal. 相似文献
4.
Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.
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C S Jones P Shankaran D J Davidson A Poulos J W Callahan 《The Biochemical journal》1983,209(2):291-297
Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100. 相似文献
5.
U1 snRNP2 isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U1 molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U1 core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U1 snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U1, U2, U4, U5 and U6 snRNPs, which contains the same four/five polypeptides as U1 snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U2, U4 and U5 were found in addition to those derived from U1. No fragment derived from U6 was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U2, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U6 snRNA, it does not contain the sequence A(U)nG which correlates well with the fact that no U6 snRNA fragment seems to resist micrococcal nuclease digestion. 相似文献
6.
M A Moreira M A Hermodson B A Larkins N C Nielsen 《The Journal of biological chemistry》1979,254(19):9921-9926
The subunits of the 11 S storage protein from soybean cultivar CX635-1-1-1 were purified and characterized. Six polypeptides with acidic isoelectric points and four with basic isoelectric points were isolated from the purified storage protein. The acidic polypeptides had phenylalanine, leucine, isoleucine, and arginine at the NH2 termini, while the basic polypeptides all had glycine at the NH2 termini. Amino acid analysis indicated that certain acidic and basic polypeptides contained 3 to 6 times more methionine than the other polypeptides. Since the low nutritional quality of legume storage proteins is due to a deficiency in methionine, this observation will have significance in efforts to improve soybean quality. The purified polypeptides were further characterized by NH2-terminal sequence analysis. Considerable homology was found between the members of individual families of acidic and basic polypeptides, indicating that the members of each family arose from a common ancestral gene. This study showed that the glycinin polypeptide composition is more complex than previous reports indicated, and for the first time characterized the various polypeptides of the 11 S storage protein by structural analysis. 相似文献
7.
Dominic W. S. Wong Sarah B. Batt Brian K. Tibbot George H. Robertson 《The protein journal》2000,19(5):373-377
Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α7-and α8-helices of the (α/β)8 domain. 相似文献
8.
Kamil Rozniakowski Andrzej Fraczyk Krystian Galecki Joanna Wietrzyk Beata Filip‐Psurska Justyna Fraczyk Zbigniew J. Kaminski Beata Kolesinska 《化学与生物多样性》2020,17(9)
Human Islet Amyloid Polypeptide (hIAPP) plays a key role in the pathogenesis of type II diabetes. The aim of this research was to search for new amyloidogenic fragments of hIAPP. An initial attempt to predict the amyloidogenic cores of polypeptides/proteins using five different computer programs did not provide conclusive results. Therefore, we synthesized hIAPP fragments covering the entire hormone. The fragments were assessed for their aggregation ability, using recommended methods to search for the amyloidogenic fragments of the polypeptides/proteins. It was found that fragments (18–22) H‐HSSNN‐OH and (33–37) H‐GSNTY‐NH2 aggregate and form stable amyloid‐like structures. Both of these fragments have a much higher antiproliferative activity relative to the RIN‐5F cell compared to the (23–27) H‐FGAIL‐OH fragment widely regarded as the amyloidogenic core of amylin. The analog of (33–37) H‐GSNTY‐NH2 containing a free carboxy group on the C‐terminal amino acid (H‐GSNTY‐OH) does not have amyloidogenic properties and can therefore be considered as a potential inhibitor of amylin aggregation. Research on the use of non‐aggregating amylin fragments as potential hormone aggregation inhibitors is ongoing. 相似文献
9.
《FEBS letters》1986,203(2):144-148
The α- and β-subunits of sweet potato mitochondrial F1ATPase were purified from the F1 complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino acid sequencing indicated that the purified β-subunit contains at least two polypeptides similar to each other. The N-terminal 18 amino acid sequence of the β-subunit showed homology to the amino acid sequence of the tobacco mitochondrial F1ATPase β-subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159-2165] between residues 56 and 73, suggesting that the N-terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting. 相似文献
10.
Foubert TR Bleazard JB Burritt JB Gripentrog JM Baniulis D Taylor RM Jesaitis AJ 《The Journal of biological chemistry》2001,276(42):38852-38861
A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation. 相似文献
11.
Summary Rice glutelin cDNA clones were isolated from a rice cDNA library constructed from immature endosperm. The isolated glutelin cDNA clones could be divided into two classes by restriction site polymorphism and sequence. They encoded polypeptides containing the same number of amino acids including a 24 amino acid signal peptide, a 282 amino acid acidic subunit and a 193 amino acid basic subunit. The coding regions were 95% homologous to each other at the nucleotide and amino acid levels. Southern hybridization analysis revealed that there were four or five copies of glutelin genes per haploid rice genome. 相似文献
12.
The antigenic properties of purified glycinin subunits were studied using antibodies prepared against them. Antisera against native glycinin did not react with the isolated subunits, and antibodies prepared against the purified subunits were not active against native glycinin. When native glycinin -was denatured, the antiglycinin immunoglobulins lost their ability to react with it, although the denatured complex was then recognized by antibodies against the purified subunits. Substantial structural rearrangement apparently occurred when the native complex was denatured and disaggregated. Acidic polypeptides A1a, A1b, and A2 had similar determinants as judged by their reactions against A1a and A1a antisera. The reaction of the A3 polypeptides with these antibodies was of lower intensity and in each case clear spurs of cross-reactivity were visible. No cross-reaction was detected between polypeptide A4 and either anti-A1a or A2. Anti-A3 antibodies reacted with each of the acidic polypeptides of glycinin, and distinct spurs of cross-reactivity were observed between A3 vs A1a, A3 vs A2, and A3 vs A4. B1 Antisera developed a reaction of identity between basic polypeptides B1 and B2, but reacted very weakly with B3 and B4. The acidic and basic polypeptides of glycinin were immunologically unrelated. The results demonstrated that immunological tests would successfully differentiate some members of the family of acidic subunits, and other immunoglobulins would discriminate between members of the family of basic subunits. 相似文献
13.
花生种子贮藏蛋白的氨基酸组成分析及发育过程中17.5 kDa多肽的合成规律 总被引:2,自引:0,他引:2
分析了两种不同蛋白质组成类型花生的子叶总蛋白3个主要组分及高甲硫氨酸类型花生的60.5、41、38.5、18和17.5kDa多肽的氨基酸组成,结果表明它们均含有17种氨基酸,其中天冬氨酸、谷氨酸和精氨酸含量最高,而甲硫氨酸含量和半胱氨酸水平都极低。高甲硫氨酸类型品种的各组分的甲硫氨酸含量均显著高于低甲硫氨酸类型品种的对应组分的甲硫氨酸含量,在这两种类型花生中伴花生球蛋白Ⅱ都是甲硫氨酸含量最高的组分 相似文献
14.
Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus 总被引:6,自引:3,他引:3
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We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components. 相似文献
15.
Chemical studies on the isolated collagen-like and globular fragment of complement component C1q. Comparative studies on bovine and human C1q 总被引:2,自引:0,他引:2
Both the collagen-like and the globular fragments of a subcomponent C1q of the first component of bovine and human complement were highly purified by enzymic digestion followed by gel filtration. Analyses by polyacrylamide gel electrophoresis showed that the former was composed of covalently linked peptide chains with an average molecular weight of 14 000, and that the latter was composed of three non-covalently linked peptide chains each having a molecular weight of approximately 15 000. Great similarities between amino acid compositions of the globular fragments and some similarities between those of the collagen-like fragments were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each globular fragment as well as between the corresponding chains of both globular fragments. These results suggested that both the collagen-like and the globular domains on the C1q molecule remained highly conserved in its evolution. 相似文献
16.
Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L 总被引:3,自引:1,他引:2
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Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 Mr. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds. 相似文献
17.
J D Semrau A Chistoserdov J Lebron A Costello J Davagnino E Kenna A J Holmes R Finch J C Murrell M E Lidstrom 《Journal of bacteriology》1995,177(11):3071-3079
A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs. 相似文献
18.
Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene 总被引:6,自引:0,他引:6
We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42. 相似文献
19.
S W Tong 《Archives of biochemistry and biophysics》1980,203(2):780-791
The linear arrangement of the three fragments of Ca2+-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH2-terminal acetylated methionyl residue of the original peptide in the Mr = 20,000 fragment. Since both the Mr = 20,000 and 30,000 polypeptides originate from a Mr = 55,000 fragment which is distinct from the Mr = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The Mr = 20,000 fragment was further cleaved by cyanogen bromide to yield a Mr = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH2-terminal portion is rich in glutamyl residues. The COOH-terminus of the Mr = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca2+-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the Mr = 30,000 fragment and hence approximately 35,000 Mr from the NH2-terminus of the original Ca2+-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca2+-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds. 相似文献
20.
Yu. A. Zolotarev A. K. Dadayan V. S. Kozik E. V. Gasanov I. V. Nazimov R. Kh. Ziganshin B. V. Vaskovsky A. N. Murashov A. L. Ksenofontov O. N. Kharybin E. N. Nikolaev N. F. Myasoedov 《Russian Journal of Bioorganic Chemistry》2014,40(1):26-35
Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity. 相似文献