Azịza ngwa ngwa
A chromophore is only as good as the pocket around it. The same protonated retinylidene Schiff base absorbs at 459 nm in one mantis-shrimp opsin and at 592 nm …
Ihe nyocha NOL14 anya-acha ọbara ọbara na-egosi n'ezie
Onye na-emepụta Peptide Ose na Peptide Brouillon na ndị ọrụ ibe nyochara stomatopod opsins iri na asaa wee hụ atọ na-abanye n'ofe 600 nm, na ọtụtụ acha anụnụ anụnụ- na opsins nwere mmetụta na-acha uhie uhie na-atụgharị ma na-atụgharị n'akụkụ ndị ọzọ (Opsin panel abstract, ezigara 17 Disemba 2025). Omume dị anya na-acha uhie uhie esighị na chromophore ọhụrụ. Nke ọ bụla n'ime protein ndị a na-eji otu retinaldehyde jikọtara ya na lysine dị ka isi Schiff., na ọnọdụ protonation nke Schiff base na-ekpebi ma polyene ọ na-amịkọrọ na ihe a na-ahụ anya ma ọ bụ ultraviolet. (Nkọwa nke preprint nke chromophore retinylidene).
Kedu ka ebe protein si eme ka chromophore na-amịpụta na mpụta ya
Ihe akaebe nọọrọ onwe ya na akpa protein na-esetịpụ ụdịdị dị iche iche sitere na klaasị chromophore dị iche. Phytochromes na cyanobacteriochromes na-ejikọta bilin tetrapyrrole linear kama ịbụ retina., etinyere na cysteine echekwara na ngalaba GAF, na nchigharị anya ha bụ steeti protonation bilin na atụmatụ (Rockwell na Lagarias, Nyocha usoro phytochrome, 2006). Steeti ndị na-amị akwụkwọ ndụ ndụ na-abụkarị ndị a na-ekpochapụ na steeti na-amịpụta ọbara ọbara protonated, yabụ otu mgbagha protonation nke na-achịkwa NOL14 na-achịkwa pigmenti kemịkalụ enweghị njikọ.
Ihe owuwu kristal nke cyanobacteriochrome uhie/acha ndụ ndụ AnPixJg2 na-egosi etu akpa akpa nwere ike isi kpalie echiche ahụ.. A na-atụ aro ka ọ bụrụ protonate n'ụdị Pr ya na mgbanaka C nitrogen, ya na agbụ akụkụ Asp291 dị ka onye na-enye proton n'ihi na mmiri pyrrole dị na phytochromes adịghị., na mgbanaka D ya na-atụgharị 61° site na ụgbọ elu B/C megide 25–45° n'ụdị phytochrome Pr. (Narikawa et al., PNAS, 2012). Ndị na-ede akwụkwọ na-ejikọta mgbagha ahụ na nkenke 648 nm Pr kacha elu nke AnPixJg2 metụtara Cph1.
Gburugburu ebe obibi chromophore nwere ike imetụta nnabata na ikuku, ma ịdị ukwuu na usoro na-adabere na usoro. Ntụle Opsin na-akpali ule nke linker, saịtị akara na ọnọdụ nyocha; ha anaghị ebu amụma ihe yiri ya 100 nm mgbanwe maka peptide akpọrọ ma ọ bụ guzobe otu njikọ hydrogen dị ka naanị ihe kpatara ya.
Kedu klas chromophore ka m ga-ahọrọ maka nyocha peptide?
Họrọ fluorophore site na iji mkpali na ndakọrịta ikuku, nchapụta na matriks n'ezie, photostability, ndabere, akara kemịkalụ na mmetụta na njide. Oge mkpochapu oge mkpụrụ akụ bara uru, mana ọ bụghị naanị ya na-ekpebi ịrụ ọrụ onyonyo.
Usoro nke ndị na-ere ahịa na-emegide ikuku redder. Ihe esijiji na-acha uhie uhie nke klaasị Cy5 nwere a 250,000 M⁻¹cm⁻¹ ọnụọgụ mkpochapụ na a 0.25 mkpụrụ nke quantum na 649/664 nm na-enye ụzọ, n'otu usoro, ka 220,000 na 0.18 na 682/701 nm wee gaa 275,000 na 0.12 na 756/780 nm (Ntuziaka nhọrọ AAT Bioquest Tide Fluor, teknuzu mpempe akwụkwọ emelitere ikpeazụ Septemba 2022). Nchapụta emetụtara sitere na 100 ka 63 ka 53 gafee akara atọ ndị ahụ. AAT Bioquest na-ere ihe eji esiji ákwà ọ tụrụ, yabụ na-emeso ụkpụrụ zuru oke dị ka data nrụpụta na omume dị ka nkwupụta ahụ.
Klas agba agba, ọ bụghị ogologo ogologo, na-esetịpụ ike absorption na agba nyere. Amine-reactive BODIPY akara kacha ogologo ogologo na-abanye na 646 nm nwere ọnụọgụ mkpochapụ nke 102,000 M⁻¹cm⁻¹, na nke 625 nm onye otu usoro na 101,000 (Akwụkwọ ntuziaka Thermo Fisher Molecular Probes, Usoro BODIPY). Nke ahụ bụ ike 40% nke nnabata nke cyanine n'ogologo n'ogologo ya, na Thermo Fisher na-erekwa ihe eji esiji ákwà ndị a.
Mmetụta gburugburu ebe obibi bụ n'otu mkpebi ahụ. Fluorescein nwere pKa phenolic gbasara 6.4 na ụdị prototropic asaa, Ya mere, mgbaàmà ya na-agbaso pH, ebe BODIPY ụdịdị dị iche iche enweghị mmetụta na gburugburu ebe obibi (Lavis na Raines). Usoro nhọrọ na-esote: dozie ogologo ikuku nke ngwa gị na matriks chọrọ, were ọnụọgụ oge mkpochapụ kachasị elu dị na ogologo ogologo ahụ, wee lelee ma a na-anabata mmetụta gburugburu ebe akwa akwa na nchekwa na serum ị ga-eji n'ezie.
Ebe na otu m ga-esi tinye akara na peptide?
Njikọ Maleimide-thiol nwere ike ịba uru na nso pH na-anọpụ iche, ma conjugate kwụsie ike na-adabere na adduct, hydrolysis na mgbanwe gburugburu ebe obibi. Njikọ thioether abụghị nkwa nkwụsi ike na matriks ọ bụla na-ebelata ma ọ bụ thiol bara ụba; nwalee conjugate n'ezie.
Otu njikọ maleimide nwere ike tufuo ibu ọrụ ya 19 awa ma ọ bụ jide ya maka 337. Na pH 7.4 na 37 Celsius C n'ime 50 mM phosphate nwere 10 mM glutathione, Baldwin na Kiick tụrụ retro-Michael ọkara ndụ nke 19 ± 2 h maka otu ihe nlereanya conjugate na 337 ± 27 h maka ọzọ, na mgbanaka-emeghe ọkara ndụ nke roughly 157 ka 215 h na mgbanaka hydrolysis tupu glutathione ịma aka igbochi mgbanwe maka ụbọchị asaa (Baldwin na Kiick, Bioconjugate Chemistry, 2011). Njikọ abụọ ahụ dị iche na nhazi agbata obi nke thiol, ọ bụghị na maleimide.
Ndị a bụ ụkpụrụ-usoro ụkpụrụ, na ọnụọgụgụ anaghị ebufe na peptide ọ bụla: ọnụego mgbanwe na-adabere na thiol na ihe ndị agbata obi, ọ bụ ya mere ogige ihe atụ abụọ ahụ ji dị iche banyere 18-fold. Ịkpọ aha peptide kpọmkwem saịtị site na cysteine na-enye njikwa ọnọdụ mana ọ na-eketa mgbanwe mgbanwe kinetics.; amine-reactive chemistry site na N-terminus ma ọ bụ lysine n'agbụ n'akụkụ dị mma mana ọ na-ahapụ njikwa ọnọdụ., n'ihi na amine ọ bụla a na-enweta bụ saịtị nyocha na ngwaahịa ndị ahụ na-ekerịta otu oke. Ihe ga-esi na ya pụta bụ na cysteine-maleimide conjugate bụ nke a kara aka maka mbelata gburugburu ebe obibi dị ka. 10 mM glutathione ma ọ bụ cell lysate kwesịrị ka enyocha nkwụsi ike ya, echeghị.
Ogologo oge ole ka onye njikọ kwesịrị ịdị, na gịnị ka a ga-eji mee ya?
Ogologo njikọ na peptides akpọrọ bụ mgbanwe na ihuenyo, ọ bụghị ntọala iji bulie elu. Ihe akaebe kacha pụta ìhè na-egosi na "ogologo ka mma": n'usoro ihe mgbochi-indocyanine green conjugate, Mkpirisi PEG linkers welitere mgbakwunye ihe ncha akwa si 21.8% ka 70.9% wee gaa 85.5%, mgbe dequenching dara site na 15.1-fold na 10.2-fold and then to 6.7-fold (Sano et al., Bioconjugate Chemistry, 2013). Otu ọrụ ahụ kọrọ a 79.7% ike na-abawanye mgbe serum incubation maka otu onye na-ewu megide roughly 41% maka onye ozo. Ndị oghere dị mkpụmkpụ na-etinyekwu ihe esiji ma hapụ obere ya.
Oke oke dị adị. Ndị conjugates bụ antibodies, ọ bụghị peptides, Ya mere ọnụ ọgụgụ ndị ahụ na-egosipụta usoro karịa nkọwapụta peptide. Ebipụtara data adịghị enye peptide na-adịgide adịgide ma ọ bụ mgbanwe IC50 dị ka ọrụ nke ogologo njikọ, na enweghị ọnụọgụ ntụgharị peptide akọwapụtara na ndekọ. Were ntụzịaka ahụ dị ka echiche iji nwalee na nyocha nke gị, ọ bụghị iwu iṅomi.
Ihe mejupụtara na-esote otu mgbagha nyocha. Onye oghere PEG bụ hydrophilic ma na-eme ka agba ahụ pụọ n'elu peptide; a hydrophobic agba na nkenke Ngwakọta Peptide linker nwere ike ikpokọta megide aromatic residues na quench. Ihe njikọ dị mkpụmkpụ siri ike nwere ike ijide agba ahụ pụọ na ihu jikọtara ya na-agbakwunyeghị ogologo.
Iwu bara uru: dịgasị abụọ ma ọ bụ atọ linker ogologo na ọ dịkarịa ala abụọ mejupụtara, tụọ arụmọrụ mgbakwunye na mgbama na matriks ị ga-eji n'ezie, ma debe njikọ kacha nso nke na-ebelata ịbelata. Kọwaa ogologo na kemistri n'ụzọ doro anya na arịrịọ njikọ ka enwere ike ịsị na nsonaazụ nyocha bụ nhazi akọwapụtara.. Peptide Bond Formation
Kedu ka m ga-esi gwa ezigbo mgbama njide sitere na polarity, protonation ma ọ bụ aggregation artifact?
Mgbanwe dị iche iche abụghị ihe akaebe nke njide ruo mgbe polarity, ewepụla protonation na mkpokọta. Akara akara nwere mmetụta gburugburu gburugburu na-agbanwe ma ike yana ogologo ogologo ya na polarity mgbaze, pH na mkpokọta, na firefly luciferin na-egosi oke mmetụta ahụ: pKa phenolic ya bụ ihe 8.7, phenol na phenolate ya na-etolite fluoresce n'ogologo n'ogologo dị iche iche, na phenolate na-achịkwa na mmiri (akara fluorescent nke nwere mmetụta gburugburu ebe obibi nke peptides, Molecules, 2021). Nchọpụta nke na-egbuke egbuke mgbe peptide zutere akpụkpọ ahụ nwere ike na-akọ na gburugburu pola dị obere karịa ka ọ na-emekọrịta ihe..
Usoro nyocha nke na-esote site na ihe akaebe ahụ nwere nzọụkwụ anọ. Mbụ, Gbaa peptide akpọrọ site na otu usoro dilution na-enweghị onye mmekọ ma gosi na mgbama ahụ dị larịị. Nke abụọ, megharịa titration na pH nke abụọ: mgbama nke na-eji pH eme ihe naanị na-akọ protonation, dị ka fluorescein pKa nke gbasara 6.4 na-ebu amụma (Lavis na Raines). Nke atọ, tulee akara polarity-enweghị mmetụta na otu usoro peptide; BODIPY ụdịdị na-enweghị mmetụta na gburugburu ebe obibi, ya mere ụdị BODIPY nke na-emezughị mgbanwe ahụ na-arụrịta ụka megide usoro dabere na njide. Nke anọ, lelee ndabere itinye uche maka mbinye aka nke mkpokọta, ebe ike na-ebili na-abụghị linearly na ụdịdị dị iche iche na-agbasawanye.
Ọmụmụ luciferin na-egosikwa ihe ezigbo mmekọrịta dị na usoro a na-achịkwa nke ọma: ijikọ peptides antimicrobial akpọrọ na lipopolysaccharide mepụtara oge ngbanwe nke na-esote usoro ntụgharị., ya na mgba ọkụ ewepụtara na free vesos micellar lipopolysaccharide kama ịbụ otu mmemme jikọtara ọnụ. (Molecules). Ọkpụkpụ biphasic na nyocha nke gị kwesịrị otu nyocha ahụ tupu akpọọ ya mmekọrịta.
Kedu ngwugwu njirimara na-egosi na peptide akpọrọ bụ ihe m nyere iwu?
Ịdị ọcha site na chromatography mmiri na-arụ ọrụ dị elu anaghị ewepụta njirimara, na njirimara site na mass spectrometry anaghị eguzobe ebe akara ahụ na-anọdụ ala. Asụsụ nkọwapụta maka ikewapụta njirimara, ịdị ọcha na ike sitere na ICH Q6B, nke na-edobe njirisi nnabata maka ngwaahịa biotechnological na bayoloji (FDA, Ụlọ ọrụ na-emepụta Peptide ICH Q6B ntuziaka ibe, nakweere 1999, ugbu a dị ka nke 2020). Q6B anaghị akpọ aha conjugates peptide, ya mere ọ bụ ntụnyere otu esi ahazi nkọwapụta, Ọ bụghị iwu chọrọ maka peptide fluorescent ọkwa nyocha.
The analytical mgbagha na-agba ọsọ dị ka ndị a. HPLC nyocha na-akọ ole n'ime ihe ahụ na-apụta dị ka otu ọnụ ọgụgụ kacha elu, nke bụ nkwupụta ịdị ọcha. Mass spectrometry na-akọ ma oke ahụ dabara na peptide a na-atụ anya ya gbakwunyere agba, nke bụ nkwupụta njirimara. Ọ dịghịkwa edozi isomers ọnọdụ: amine-reactive labeling na-etinye agba na N-terminus ma ọ bụ na lysine ọ bụla nwere ike ịnweta., na ngwaahịa ndị ahụ na-ejikọta ọnụ ma na-ekerịta otu ihe na-adịghị mma. Asambodo nyocha nke depụtara naanị pasentị ịdị ọcha na oke, yabụ enweghị ike ịgwa gị ma peptide akpọpụtara ọ bụ otu ụdị ị kọwapụtara..
Otu ngwugwu na-aza ajụjụ a gụnyere chromatogram na ntinye ya, uka ụdịdị dị iche iche na ndị hụrụ na gbakọọ ọtụtụ, usoro akara na saịtị, nha-na-peptide ruru na usoro eji achọpụta ya, na ndekọ ọtụtụ ihe maka ogbe ebugara. Rịọ chromatogram karịa pasentị naanị, ma jụọ kemistri akara nke ejiri mee ihe, n'ihi na nke ahụ na-ekpebi ma ọnọdụ isomers ga-ekwe omume ma ọlị.
Na-etinye ya ọnụ: ndepụta nkọwapụta maka imepụta nyocha peptide kacha mma
Ụdị mgbanwe ise ahụ na-aghọ nkọwapụta mgbe edechara nke ọ bụla tupu etinyere iwu. Ịmepụta nyocha peptide kacha mma bụ ọzụzụ nke ịjụ ịhapụ nke ọ bụla n'ime ise ahụ na ndabara nke ndị na-ebubata ya., n'ihi na ndabara (kacha atụ ụjọ anwụ, aha nwoke, ogologo njikọ, pasent nke ịdị ọcha) bụ onye ọ bụla nwere ike ịgbachitere onwe ya na nke ọ bụla nwere ike ịmepụta nyocha enweghị nkọwa.
|
Na-agbanwe agbanwe |
Ihe ị ga-akọwapụta |
Ihe akaebe n'azụ criterion |
|---|---|---|
|
Klas chromophore |
Mkpali na mgbapụta achọrọ, mgbe ahụ ọnụọgụ mkpochapụ kacha elu × quantum na-amịpụta n'ogologo nfe ahụ, gbakwunyere nkwupụta nke mmetụta gburugburu ebe obibi |
Icha ọkụ bụ ε × Φ (Lavis na Raines); Mkpụrụ cyanine na-acha uhie uhie dị anya na-ada ka ikuku na-akpali uhie (Ọnụ ego nke AAT Bioquest) Ụlọ ọrụ Peptides |
|
Mgbakwunye kemịkalụ na saịtị |
Cysteine-maleimide ma ọ bụ amine-reactive, kpọmkwem ihe fọdụrụ, na ihe nchekwa nke conjugate ga-ezute |
pH 6.5-7.5 windo na reductant ndakọrịta (Thermo Fisher); 19 h megide 337 h gbanwere ọkara ndụ (Baldwin na Kiick) |
|
Njikọ |
Chemistry na ogologo, nwere ogologo abụọ e hotara ma ọ bụrụ na nyocha ahụ dị ọhụrụ |
Mgbakwunye na mwepu mgbanwe na ogologo njikọ na mgbochi mgbochi mmadụ (Sano et al.) |
|
gburugburu ebe obibi |
Nyochaa pH, Serum ma ọ bụ glutathione itinye uche, na akara njikwa enweghị mmetụta polarity |
Akara akara nwere mmetụta gburugburu na-eji polarity na pH gbanwere (Molecules) |
|
Njirimara |
Chromatogram na ntinye, uka ụdịdị dị iche iche na hụrụ na gbakọọ uka, saịtị ịkpọ aha, nha-na-peptide ruru, ọtụtụ ndekọ |
njirimara, ịdị ọcha na ike bụ ụdị nkọwapụta dị iche iche (I Q6B) |
Ahịrị abụọ kwesịrị ahịrịokwu nke ọ bụla. Na mgbakwunye, rịọ maka nkwụsi ike nke conjugate na gburugburu ebe obibi mbelata nke nyocha, n'ihi na kinetics ihe nlereanya na-egosi usoro-nke-n'ịdị ukwuu gbasaa n'etiti thiols abụọ n'otu nchekwa.. Na njirimara, Rịọ maka saịtị akara na ederede, n'ihi na conjugate nke enwetara n'ọha na saịtị ekwughị okwu dakọtara na ọtụtụ isomers ọnọdụ.
Otu ahịrị ise ahụ na-arụ ọrụ dị ka akwụkwọ nyocha maka onye mmekọ njikọ. Onye na-ebubata ihe nwere ike ịza ajụjụ chromophore na ọnụọgụ mkpochapụ yana mkpụrụ quantum, ajụjụ mgbakwunye na kemistri na saịtị, ajụjụ njikọ ahụ nwere usoro akọwapụtara, ajụjụ gburugburu ebe obibi nwere nkwupụta kwụsie ike, na ajụjụ njiri mara ya na chromatogram na ọtụtụ ụdị dị iche iche na-aza na ọkwa nke nyocha chọrọ.. Onye na-ebubata ihe na-aza naanị ajụjụ ogologo ogologo anabatala nke kacha nwee ihe ọmụma.
Ajụjụ a na-ajụkarị
Fluorophore na-acha uhie uhie ọ dị mma mgbe niile maka nyocha peptide?
Mba. Icha ọkụ bụ ngwaahịa nke nbibi na mkpụrụ nke quantum, ya mere agba agba ogologo ogologo nke nwere obere mkpụrụ nke quantum nwere ike ịdị ntakịrị karịa nke dị mkpụmkpụ-ohere ogologo. (Lavis na Raines). N'otu usoro cyanine na-acha uhie uhie, ihie ihie dara ka mpụta ahụ kpaliri uhie, si 649/664 nm na 682/701 nm na 756/780 nm (Ọnụ ego nke AAT Bioquest).
Kedu ihe bụ ntinye peptide kpọmkwem saịtị, na mgbe ọ dị mkpa?
Ịkpọ aha saịtị akọwapụtara na-edowe agba ahụ n'otu ihe fọdụrụ akọwapụtara, na-abụkarị cysteine site na maleimide ma ọ bụ aka thiol-reactive, karịa na nke ọ bụla amine na-emeghachi omume mbụ. Ọ dị mkpa mgbe isomers ọnọdụ ga-agbanwe nyocha ahụ, na ọ na-ebu ọnụ kinetics: maleimide-thiol gbanwere ọkara ndụ sitere na 19 ± 2 h na 337 ± 27 h n'etiti ihe atụ abụọ conjugates na pH 7.4 na 37 °C na 10 mM glutathione (Baldwin na Kiick).
Kedu ihe kwesịrị ịgụnye asambodo nyocha maka peptide akpọrọ?
Opekempe chromatogram nwere njikọta, a uka ụdịdị dị iche iche na hụrụ na gbakọọ ọtụtụ, kemịkalụ labeling na saịtị, nha-na-peptide ruru na usoro ya, na ndekọ ndị akọwapụtara nke ọma. Ịdị ọcha na njirimara bụ nkwupụta dị iche iche, na ọ dịghịkwa na-edozi ọnọdụ isomers si amine-reactive labeling. ICH Q6B bụ ihe atụ maka ikewapụta njirimara, ịdị ọcha na ike, ọ bụ ezie na ọ naghị akpọ peptide conjugates (FDA).
Mmechi
Banyere onye nkwusa. MOL Mgbanwe bụ nyocha na nzukọ mmepe peptide na-ere nhazi peptide omenala na ọrụ mgbanwe, gụnyere akara fluorescent, ya mere ọ nwere mmasị azụmahịa na otu esi eme mkpebi ịde akara. Buru nke ahụ n'uche tụọ echiche ya; ntuziaka dị n'elu anaghị adabere na ọrụ ya.
