RESUMEN
Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon ß-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.
Asunto(s)
Interferón beta-1a/química , Polietilenglicoles/química , Cromatografía por Intercambio Iónico , Glicoproteínas/química , Glicosilación , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem/métodosRESUMEN
Photochemical crosslinking is now a powerful method for studying protein-nucleic acid interactions. UV light is a zero-length crosslinking agent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidence for close protein-nucleic acid interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours are necessary. Such prolonged irradiation allows for the artifactual redistribution of proteins and precludes kinetic studies. The use of UV lasers overcomes these difficulties since the number of photons required for the crosslinking may be delivered in time intervals on the order of nano- or even picoseconds. We described detailed procedures for UV laser-induced protein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of covalently crosslinked protein-DNA complexes, immunochemical techniques for both the identification and isolation of specific protein-DNA complexes and the identification of the crosslinked DNA sequences, are reviewed in detail. The application of UV laser crosslinking in kinetic studies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Animales , Secuencia de Bases , Núcleo Celular/ultraestructura , Centrifugación por Gradiente de Densidad/métodos , Reactivos de Enlaces Cruzados , ADN/química , ADN/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Indicadores y Reactivos , Cinética , Rayos Láser , Regiones Promotoras Genéticas , TATA Box , Proteína de Unión a TATA-Box , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificación , Rayos UltravioletaRESUMEN
Bis(tert-butyldimethylsiloxy)- (7), bis(dimethylthexylsiloxy)- (8), bis(tri-n-hexylsiloxy)- (9), and bis(dimethyloctadecylsiloxy)silicon 2,3-naphthalocyanines (10) were prepared via substitution of the bis(hydroxy) precursor with the corresponding chlorosilane ligands and characterized by spectroscopic and combustion analyses. They show strong absorption around 780 nm where tissues exhibit optimal transparency. Compounds 7-10 are capable of producing singlet oxygen. They are relatively photostable although less stable than the analogous phthalocyanine, i.e., the bis-(dimethylthexylsiloxy)silicon phthalocyanine (12). They were evaluated as potential photosensitizers for the photodynamic therapy (PDT) of cancer in vitro against V-79 cells and in vivo against the EMT-6 tumor in Balb/c mice. In vitro all four dyes showed limited phototoxicity combined with substantial dark toxicity. Surprisingly, in vivo (i.v., 0.1 mumol/kg, 24 h prior to the photoirradiation of the tumor with 780-nm light, 190 mW/cm2, 400 J/cm2) all dyes induced tumor regression in at least 50% of mice whereas compound 8 gave a complete tumor response in 80% of mice without apparent systemic toxicity at doses as high as 10 mumol/kg. At 24 h postinjection, compound 8 showed a favorable tumor to muscle ratio of 7, assuring minimal damage to the healthy tissue surrounding the tumor during PDT. Our data confirm the potential of silicon naphthalocyanines as far-red-shifted photosensitizers for the PDT of cancer and indicate the importance of the selection of the two axial silicon ligands for optimal photodynamic efficacy.
Asunto(s)
Metaloporfirinas/síntesis química , Compuestos de Organosilicio/síntesis química , Fármacos Fotosensibilizantes/síntesis química , Animales , Antineoplásicos/uso terapéutico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Estabilidad de Medicamentos , Espectroscopía de Resonancia Magnética , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metaloporfirinas/farmacocinética , Metaloporfirinas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Trasplante de Neoplasias , Compuestos de Organosilicio/farmacocinética , Compuestos de Organosilicio/uso terapéutico , Fotoquímica , Fármacos Fotosensibilizantes/uso terapéutico , EspectrofotometríaRESUMEN
A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.
Asunto(s)
Carboxihemoglobina/efectos de la radiación , Adulto , Carboxihemoglobina/metabolismo , Hemoglobina A/aislamiento & purificación , Humanos , Cinética , Rayos Láser , Ligandos , Fotólisis , Conformación Proteica , Espectrometría Raman/métodos , Factores de TiempoRESUMEN
The role of the topoisomerase enzyme in DNA recombination was investigated by extracting chromosomal deoxyribonucleoproteins from a variety of cultured mammalian cells and assaying for the formation of recombinant DNA structures. Although each of the crude deoxyribonucleoprotein preparations contained topoisomerase activity, they did not all contain DNA-recombining activity. A distinct, perhaps novel, enzyme may therefore promote DNA recombination in these cell-free systems.