This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

NOTA: Os baços foram derivadas de ratinhos (6-10 semanas de idade) e todas as experiências com animais foram realizadas de acordo com as orientações éticas animais de MacCallum Cancer Centre, Peter (E486). 1. Preparação de Amostras. <ol><li> Preparação das células de rato NK ativadas. <ol><li> Isolar células NK ingênuas primários de uma única célula suspensões dos baços de C57BL / 6 ou B6.GrzmB - / - (null gene GzmB) ratos por seleção negativa usando kits disponíveis no mercado. </li><li> Siga o protocolo dos fabricantes. Resumidamente, magneticamente marcar células NK &quot;não&quot;, tais como as células T e B, células dendríticas, granulócitos, macrófagos e células vermelhas do sangue, utilizando anticorpos conjugados com biotina contra os seus marcadores de superfície específicos. Use separação magnética com contas anti-biotina para destruir as células &quot;não-NK&quot;. </li><li> Cultura isolada células NK durante 5-7 dias em meio contendo IL-2 (1000 U / ml) a 700.000 células / ml em placas de 24 poçosa 37 &#160; ° C. </li><li> Alternativamente, as células T activadas utilização 19, antígeno-primária de CTL restrito a partir do receptor de células T OT1 ratinhos transgénicos 20, os CTL gerados em culturas de linfócitos mistas, 21 ou sobrenadantes de células NK activadas para determinar a actividade de secretada GzmB 19. </li></ol></li><li> Manutenção da linha de células NK humana e o isolamento de células NK primárias. <ol><li> Manter leucemia NK humana (células YT) no Roswell Park Memorial Institute (RPMI) meio suplementado com 10% de soro fetal de vitelo. Em alternativa, purificar as células NK primárias a partir do sangue periférico de indivíduos saudáveis ​​e estimular durante 2-3 dias em meio contendo IL-2 a 100 U / ml como previamente descrito 22. </li></ol></li><li> Geração de lisados ​​celulares. <ol><li> Lave as células três vezes em PBS (pH 7,4), seguida de suspensão em P-40 tampão de lise Nonidet (0,1% de NP-40, NaCl 250 mM, Hepes 25 mM, EDTA 2,5 mM). Idealmente, lisar um mínimo de 5 x 10 6 </saté&gt; células NK, ou 1 x 10 7 células T em 100 ul de tampão. Não adicionar inibidores da protease, como muitos são inibidores irreversíveis das proteases de serina, e em particular, tais como triptases GzmA. Incubar em gelo durante 20 min, e em seguida sedimentar os núcleos numa microcentrifugadora a 15.000 xg durante 10 min. Descartar o sedimento nuclear e transferir o sobrenadante para um tubo fresco. </li></ol></li><li> Determinar a concentração de proteína dos lisados. <ol><li> Usar um protocolo disponível comercialmente de escolha, tal como, Bradford ou ácido bicinconínico (BCA) e determinar a concentração de proteína. Certifique-se de que a concentração é de aproximadamente 2 mg / ml mínimo. Armazenar em lisados ​​de -20 ° C até ser necessário (granzima actividade é estável durante vários meses a -20 ° C). </li></ol></li></ol> 2. A granzima B Ensaio de Actividade <ol><li> Preparação dos Reagentes <ol><li> Preparar um estoque 10 mM dos substratos (Boc-AAD-S-Bzl ou Ac-IEPD-pNA) em DMSO (~ 5 mg / ml) e armazenam-se em aliquotas a -20 &amp; #176; C. Prepara-se uma diluição de trabalho acabado e descartar depois de cada dia, como Boc-AAD-S-Bzl hidrolisa parcialmente em armazenamento em DMSO. </li><li> Preparar um estoque 250 mM do reagente colorimétrico (5,5&#39;-ditio-bis (ácido 2-nitrobenzóico), DTNB) em DMSO (0,09 g / ml) e armazenar a -20 ° C. Prepare uma diluição de trabalho de fresco todos os dias. Este reagente é necessária apenas para substratos com grupos de indicadores SBzl, não pNA. </li><li> Make up tampão fresco (0,1 M HEPES, 0,05 M MgCl2, pH 7,3) a cada 2-3 semanas. </li></ol></li><li> Traga o DTNB e substratos sintéticos para a temperatura ambiente. Diluir, substratos (1:16, por exemplo, 100 ml em 1,6 ml de tampão) e DTNB (1: 250, por exemplo, 10 jul em 2,5 ml de tampão) e misture bem. </li><li> Dilui-se um mínimo de 100 ug de proteína de lisado de cada amostra em tampão para um volume final de 160 ul. Usando uma pipeta multicanal adicionar 50 ul por poço em triplicado (~ 33,3 ng / cavidade) em um &#39;&#39; U &#39;&#39; vinilo inferior placa de 96 poços.Incluir um &quot;tampão-only&quot; controle (também em triplicado). </li><li> Adicione 100 ml de diluída DTNB para o controle samples / tampão (omitir esta etapa quando se trabalha com Ac-IEPD-pNA. NOTA: A clivagem de Ac-IEPD-pNA por GzmB liberta fluorogénico directamente pNA (Figura 4B). </li><li> Usar uma pipeta de canais múltiplos para adicionar rapidamente 50 ul de substrato diluído para cada conjunto de triplicados, começando com o tampão, em seguida, todos os controlos negativos e terminando com amostras positivas esperadas. </li><li> Colocar a placa no leitor de placas. Medir a actividade Asp-ase por hidrólise de Boc-Ala-Ala-Asp-S-Bzl / Ac-IEPD-pNA em um leitor de microplacas a OD 405 nm. Use um protocolo de ensaio cinético, levar 25 leituras, a cada 15 segundos (cerca de 6 min de tempo de leitura total). </li><li> Use as leituras de absorvância gerado em cada ponto de tempo para calcular manualmente a taxa de clivagem do substrato caso o software do leitor de placas não podem gerar um valor de Vmax. </li></ol> 3. Análise de Data NOTA: Os dados gerados são apresentados como a velocidade máxima, definida como a mudança de OD ao longo do tempo (mOD / min). <ol><li> Para analisar os dados, determinar o valor máximo de clivagem do substrato, o qual é calculado a partir da taxa de variação de absorvância. Faça isso usando automaticamente o software leitor de placas, que normalmente tem essa opção. </li><li> Como alternativa, depois de ver as parcelas cinéticos de variação de absorvância, que são gerados durante o ensaio de cinética no leitor de placa, manualmente selecionar os pontos de dados que dão a mais íngreme linha reta. Para a maioria dos ensaios, a taxa máxima é obtida dentro da primeira 2 minutos de ensaio. </li><li> Subtraia a leitura OD inicial da maior leitura OD nesta linha reta e dividir pelo intervalo de tempo. Transferência de dados brutos para o Microsoft Excel / Gráfico pad para análise estatística e display gráfico. </li></ol>

<ol>
	<li>Trapani, J. A., Browne, K. A., Dawson, M., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+functional+Asp-ase+(natural+killer+cell+granzyme+B)+using+a+monoclonal+antibody).">Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody).</a> Biochem Biophys Res Commun. 195 (2), 910-920 (1993).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell Death Differ. 19 (1), 28-35 (2012).</li><li>Hoves, S., Trapani, J. A., Voskoboinik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+battlefield+of+perforin/granzyme+cell+death+pathways.">The battlefield of perforin/granzyme cell death pathways.</a> J Leukoc Biol. , (2009).</li><li>Peters, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte+granules+are+secretory+lysosomes,+containing+both+perforin+and+granzymes.">Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.</a> J Exp Med. 173 (5), 1099-1109 (1991).</li><li>Berthou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granzyme+B+and+Fas+ligand+proteins+by+human+keratinocytes+contributes+to+epidermal+cell+defense.">Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.</a> J Immunol. 159 (11), 5293-5300 (1997).</li><li>Tschopp, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B,+a+novel+mediator+of+allergic+inflammation:+its+induction+and+release+in+blood+basophils+and+human+asthma.">Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma.</a> Blood. 108 (7), 2290-2299 (2006).</li><li>Strik, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mast+cells+produce+and+release+the+cytotoxic+lymphocyte+associated+protease+granzyme+B+upon+activation.">Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.</a> Mol Immunol. 44 (14), 3462-3472 (2007).</li><li>Jahrsdorfer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+produced+by+human+plasmacytoid+dendritic+cells+suppresses+T+cell+expansion.">Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion.</a> Blood. , (2009).</li><li>Hagn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cells+secrete+granzyme+B+when+recognizing+viral+antigens+in+the+context+of+the+acute+phase+cytokine+IL-21.">Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21.</a> J Immunol. 183 (3), 1838-1845 (2009).</li><li>Hagn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cells+differentiate+into+granzyme+B-secreting+cytotoxic+B+lymphocytes+upon+incomplete+T-cell+help.">Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help.</a> Immunol Cell Biol. 90 (4), 457-467 (2012).</li><li>Froelich, C. J., Pardo, J., Simon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule-associated+serine+proteases:+granzymes+might+not+just+be+killer+proteases.">Granule-associated serine proteases: granzymes might not just be killer proteases.</a> Trends Immunol. 30 (3), 117-123 (2009).</li><li>Hagn, M., Jahrsdorfer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+human+B+cells+secrete+granzyme+B?+Insights+into+a+novel+B-cell+differentiation+pathway.">Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.</a> Oncoimmunology. 1 (8), 1368-1375 (2012).</li><li>Susanto, O., Trapani, J. A., Brasacchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+granzyme+biology.">Controversies in granzyme biology.</a> Tissue Antigens. 80 (6), 477-487 (2012).</li><li>Walch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157 (6), 1309-1323 (2014).</li><li>Odake, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+cytotoxic+T+lymphocyte+serine+proteases:+subsite+mapping+with+peptide+thioester+substrates+and+inhibition+of+enzyme+activity+and+cytolysis+by+isocoumarins.">Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.</a> Biochemistry. 30 (8), 2217-2227 (1991).</li><li>Casciola-Rosen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+human+granzyme+B+have+distinct+tetrapeptide+specificities+and+abilities+to+recruit+the+bid+pathway.">Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway.</a> J Biol Chem. 282 (7), 4545-4552 (2007).</li><li>Kaiserman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+human+and+mouse+granzymes+are+structurally+and+functionally+divergent.">The major human and mouse granzymes are structurally and functionally divergent.</a> J Cell Biol. 175 (4), 619-630 (2006).</li><li>Powers, J. C., Kam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+thioester+substrates+for+serine+peptidases+and+metalloendopeptidases.">Peptide thioester substrates for serine peptidases and metalloendopeptidases.</a> Methods Enzymol. 248, 3-18 (1995).</li><li>Hagn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+mouse+B+cells+lack+expression+of+granzyme.">Activated mouse B cells lack expression of granzyme.</a> B. J Immunol. 188 (2), 3886-3892 (2012).</li><li>Konjar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+mouse+perforin+are+processed+in+part+through+cleavage+by+the+lysosomal+cysteine+proteinase+cathepsin+L.">Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L.</a> Immunology. 131 (2), 257-267 (2010).</li><li>Sutton, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+active+granzyme+B+in+cathepsin+C-null+lymphocytes+is+sufficient+for+perforin-dependent+target+cell+apoptosis.">Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.</a> J Cell Biol. 176 (4), 425-433 (2007).</li><li>Trapani, J. A., Smyth, M. J., Apostolidis, V. A., Dawson, M., Browne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule+serine+proteases+are+normal+nuclear+constituents+of+natural+killer+cells.">Granule serine proteases are normal nuclear constituents of natural killer cells.</a> J Biol Chem. 269 (28), 18359-18365 (1994).</li><li>Cullen, S. P., Adrain, C., Luthi, A. U., Duriez, P. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+granzyme+B+exhibit+divergent+substrate+preferences.">Human and murine granzyme B exhibit divergent substrate preferences.</a> J Cell Biol. 176 (4), 435-444 (2007).</li><li>Thornberry, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+approach+defines+specificities+of+members+of+the+caspase+family+and+granzyme+B.+Functional+relationships+established+for+key+mediators+of+apoptosis.">A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.</a> J Biol Chem. 272 (29), 17907-17911 (1997).</li><li>Bird, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+regulation+of+apoptosis:+the+cytotoxic+lymphocyte+serpin+proteinase+inhibitor+9+protects+against+granzyme+B-mediated+apoptosis+without+perturbing+the+Fas+cell+death+pathway.">Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.</a> Mol Cell Biol. 18 (11), 6387-6398 (1998).</li><li>Bots, M., Medema, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serpins+in+T+cell+immunity.">Serpins in T cell immunity.</a> J Leukoc Biol. 84 (5), 1238-1247 (2008).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+10+murine+ovalbumin+serpins+includes+two+homologs+of+proteinase+inhibitor+8+and+two+homologs+of+the+granzyme+B+inhibitor+(proteinase+inhibitor+9).">A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).</a> J Biol Chem. 272 (24), 15434-15441 (1997).</li><li>Bird, C. H., Hitchen, C., Prescott, M., Harper, I., Bird, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+granzyme+B+tissue+distribution+and+cellular+localisation.">Immunodetection of granzyme B tissue distribution and cellular localisation.</a> Methods Mol Biol. 844, 237-250 (2012).</li><li>Jenkins, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+CTL+activity+for+different+CD8++effector+T+cells+supports+the+idea+that+lower+TCR/epitope+avidity+may+be+advantageous+for+target+cell+killing.">Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.</a> Cell Death Differ. 16 (4), 537-542 (2009).</li><li>Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotoxic+T+cell+granule+serine+protease+granzyme+H+has+chymotrypsin-like+(chymase)+activity+and+is+taken+up+into+cytoplasmic+vesicles+reminiscent+of+granzyme+B-containing+endosomes.">The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.</a> J Biol Chem. 274 (43), 30468-30473 (1999).</li><li>Sutton, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+apoptosis+by+granzyme+B+requires+direct+cleavage+of+bid,+but+not+direct+granzyme+B-mediated+caspase+activation.">Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.</a> J Exp Med. 192 (10), 1403-1414 (2000).</li></ol>

The authors have nothing to disclose.

Historicamente os granzimas foram identificados como moléculas efectoras principais de linfócitos citotóxicos (CTL e as células NK), capazes de induzir uma rápida morte apoptótica em células alvo. Isto foi devido principalmente à ação de GzmB, que clivada moléculas de substrato-alvo em aspartato (D) os resíduos e, assim, foi capaz de ativar a cascata caspase por ambas clivagem pró-caspases, assim como vários de seus alvos a jusante. No entanto, é agora apreciado que a expressão GzmB não está confinada ...

Granzimas são uma família de serina-proteases encontradas nos lisossomas secretoras de células assassinas naturais (NK) e linfócitos T citotóxicos (CTL) 1. Cinco granzimas diferentes existem em seres humanos (A, B, H, K e M), e dez ratos (A - G, K, M e N) 2,3. Granzyme A e B Granzyme (GzmA, GzmB) são os mais abundantes e têm sido amplamente estudados no cenário humano e roedores. A função clássica de GzmB é a indução de apoptose em células alvo executadas em conjunto com a proteína perforina de formação de poros, o que permite a granzima para aceder ao citosol da célula alvo 4. Embora a expressão GzmB é inequivocamente encontrada em linfócitos citotóxicos, os estudos recentes têm-se debruçado sobre uma variedade de outros tipos de células que expressam GzmB, incluindo mas não se limitando aos queratinócitos 5, 6 basófilos, mastócitos, células dendríticas 7 plasmocitoides 8, 9 e células B, 10.Neste contexto, as funções GzmB não apoptóticas foram revelados desde a participação em processos inflamatórios, remodelação de tecidos e outras propriedades imunomoduladoras 11-14. Tendo em conta que um papel biológico mais amplo tem sido proposto para GzmB do que se suspeitava anteriormente, os pesquisadores necessitam de ferramentas confiáveis ​​e específicos para a sua detecção. De vantagem é requisito específico da GzmB para clivar no lado carboxilo dos resíduos de ácido aspártico, uma propriedade únicas entre as proteases de serina 15 eucarióticas. Rato, humano e de rato GzmB são estruturalmente muito semelhantes, no entanto, a especificidade do substrato prolongado de rato GzmB subtilmente diferente daquele de ambos humano e de rato 16, o que significa que certos substratos genéricos com Asp no terminal (P1) pode ser clivado eficazmente por GzmB de todas as três espécies, enquanto que outros substratos com sequências mais complicadas montante de P1 pode dar resultados muito variáveis. Tanto no passado e li mais recenteterature, este fato tem causado confusão e má interpretação do significado biológico de alguns achados experimentais considerável, embora cuidadosamente controlados, estudos cinéticos têm procurado corrigir a situação 17. Neste trabalho procuramos ilustrar esses pontos usando dois substratos disponíveis no mercado, ou seja, Boc-Ala-Ala-Asp-SBzl e N-acetil-Ile-Glu-Pro-Asp-p-nitroanilida. Os dois reagentes fazer gerar diferentes grupos reativos após a clivagem (a sulfidrila livre versus um paranitroanilida livre fluorescente), mas isso não tem nenhum efeito sobre a clivagem proteolítica. O protocolo descrito é uma adaptação moderna de um protocolo muito velho 18, mas deve ajudar os pesquisadores a utilizar os diferentes substratos GzmB adequadamente, além de fornecer um quadro metodológico para detectar a atividade de outros granzimas, como GzmA e GzmH.

<table border="0" cellpadding="0" cellspacing="0" width="1133">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Product</td>
			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

O substrato Boc-AAD-S-Bzl é específico para GzmB O GzmB protease serina é um dos principais constituintes de linfócitos citotóxicos (CTL e as células NK) e é predominantemente responsável pela indução da morte apoptótica rápida em células alvo, tais como células transformadas ou infectadas com vírus. Isto é, em grande parte devido à sua preferência de substrato para a clivagem após certos resíduos de aspartato específicas em proteínas selecionadas, um a...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Um ensaio colorimétrico que mede Especificamente granzima B Actividade proteolítica: Hidrólise de Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Research

JoVE Journal

Chemistry

12.9K visualizações.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Vídeo: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Rápidas de teste colorimétrico para Qualitativamente Distinguir RNA e DNA em amostras biomoleculares

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Química

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Steady-state Pré-steady-state, e Single-turnover Medição, Kinetic para DNA glicosilase Atividade

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Monitoramento baseado em fluorescência de PAD4 Atividade através de um substrato Analog Pro-fluorescência

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

Dinâmica eletroquímico Medição de íons cloreto

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

Um Método para Manipular a Tensão Superficial de um Metal Líquido via Oxidação e Redução Superficial

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

Preparação e Reatividade de uma Triphosphenium Brometo Sal: Uma fonte adequada e estável de fósforo (I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Medição simultânea de HDAC1 e atividade HDAC6 em HeLa células usando UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

Microfluidic preparação dos atuadores do líquido cristalino de elastômero

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

Síntese de Ésteres Via Esterificação de Steglich Mais Sustentável em Acetonitrila

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

Ensaios de atividade com base em RMN para determinação da inibição de compostos, valores de IC50 , atividade Artifactual e atividade de células inteiras de Ribohidrolases nucleósidos

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...