| Abstract | Selektivno presijecanje peptidne okosnice jedna je od najvećih prepreka prilikom analize proteina. Peptidna veza je stabilizirana rezonantnom strukturom i kao takva izuzetno nepodložna hidrolizi. Dostupni reagensi za cijepanje često djeluju neselektivno, neki su i otrovni te zahtijevaju visok utrošak i ograničene uvjete reakcije. Ovi razlozi potiču razvoj novih i učinkovitijih proteaza.
Hidroliza peptidne veze potpomognuta metalnim ionima je obećavajuća alternativa enzimskom cijepanju proteina s perspektivnim primjenama u biokemiji i bioinženjerstvu. Mnogi ioni i kompleksi metala testirani su na takvu reaktivnost na većem broju supstrata, od dipeptida i oligopeptida do proteina. Među takvim, visoku učinkovitost pokazali su prijelazni metali.
U ovom radu ispitana je regioselektivnost presijecanja peptidne veze u sekvenciji goveđeg hemoglobina i humanog monoklonskog antitijela IgG1 različitim kompleksima platine(II) i paladija(II) kako bi odredili na koji način ovi metalni ioni i sterički faktori usmjeravaju proteolizu.
Selektivna hidroliza peptidne veze postignuta je tijekom 12-satne inkubacije, pri pH 2.5 i temperaturi 40 ᵒC te primjenom kompleksa u suvišku 10:1 spram mjesta vezanja na supstratu. Nastali peptidni fragmenti određeni su tandemskom spektrometrijom masa na temelju specifičnog fragmentacijskog uzorka prekursorskog iona. Pritom je kompleks platine(II) s bidentatno koordiniranim etilendiaminom pokazao veću selektivnost naspram analoga paladija(II). Za spoj [Pt(en)(H2O)2]2+ identificirano je 17 fragmenata u sekvenciji hemoglobina, odnosno 10 fragmenata za IgG1. U slučaju kompleksa [Pd(en)(H2O)2]2+, određen je slijed 34 peptida u sekvenciji hemoglobina te 11 peptida u sekvenciji IgG1.
Mjesta presijecanja, utvrđena su analizom aminokiselina u neposrednoj blizini, a to su: l-metionin, l-cistein te l-histidin. U manjoj mjeri hidroliza goveđeg hemoglobina dogodila se u blizini l-asparaginske kiseline (hemoglobin) te nepolarnih l-glicina i l-triptofana (IgG1). Kada je ispitivanje provedeno sa složenim, dinuklearnim spojevima tipa [{M(en)(H2O)}2(-L)]4+, premoštenim aromatskim pirazinom i piridazinom, učinkovitost i prinos hidrolize bili su gotovo jednaki. Naši rezultati ukazuju na mogućnost primjene spojeva platine(II) i paladija(II) za proteolizu, prikazuju uvjete potrebne za reakciju te selektivnost proteolize kao temelj primjene ovih spojeva u biokemijskoj praksi. |
| Abstract (english) | Selective cleavage of the peptide backbone is one of the major obstacles in protein analysis. Peptide bond is stabilized by a resonant structure, and therefore extremely insusceptible to hydrolysis. Accessible cleavage reagents are often nonselective, some are toxic, require high fold molar excess and harsh conditions. Aforementioned reasons prompted the development of new, more efficient chemical proteases.
Metal ion-assisted peptide bond hydrolysis is a favourable alternative for enzymatic cleavage of proteins with promising applications in biochemistry and bioengineering. Many metal ions and their complexes have been tested for such reactivity on a number of substrates, from dipeptides and oligopeptides to proteins. Among such, high efficiency was demonstrated by transition metal ions.
In this study, we investigated the regioselectivity of peptide bond cleavage by various platinum(II) and palladium(II) complexes to determine how these metal ions, and steric factors direct proteolysis of bovine hemoglobin and human monoclonal antibody IgG1.
Selective hydrolysis was accomplished by applying 10-fold molar excess of reagents, during 12-hour incubation, at pH 2.5 and 40 ᵒC. Product fragments were determined, based on the specific fragmentation pattern of their precursor ion, by tandem mass spectrometry. Thereat, platinum(II) complex with bidentate-coordinated ethylenediamine showed greater selectivity than the palladium(II) analog. For [Pt(en)(H2O)2]2+ 17 fragments were identified after cleaving hemoglobin sequence, apropos to 10 fragments for IgG1. In respect to [Pd(en)(H2O)2]2+, we assigned 34 product fragments to hemoglobin polypeptide chain, and 11 fragments to the sequence of IgG1.
Regioselectivity of cleavage, determined by amino acids in the immediate vicinity, included side chains of l-methionine, l-cysteine and l-histidine. To a lesser extent, hydrolysis of bovine hemoglobin occurred near l-aspartic
acid (hemoglobin) and non-polar l-glycine and l-tryptophan (IgG1). When we examined efficiency of more complex, dinuclear compounds bridged with aromatic pyrazine or pyridazine, here shown as [{M(en)(H2O)}2(-L)]4+, the efficacy and yield of hydrolysis were almost equal. Our results indicate the possibility of using platinum(II) and palladium(II) compounds for proteolysis, show reaction conditions in which hydrolysis occurs, and demonstrate selectivity of proteolysis as the basis for the application of these compounds in biochemical practice. |
