Methylene Carbene Labeling Of Proteins

GÓMEZ G.E; DELFINO J.M

Angra do Reis, Brasil.

Congreso; 1st Latin American Protein Society Meeting.; 2004

METHYLENE CARBENE LABELING OF PROTEINS Gómez, G.E., Delfino, J.M. IQUIFIB (Universidad de Buenos Aires-CONICET), Junín 956, 1113 Buenos Aires, Argentina. E-mails: ggomez@qb.ffyb.uba.ar, delfino@qb.ffyb.uba.ar Diazirine (DZN) is a photoreactive gas similar in size to water. By UV irradiation it generates methylene carbene (:CH2), which reacts unselectively with its molecular cage, inserting even into C-H bonds. 3H-DZN has been successfully used in our laboratory for studying protein structure and folding –as in bovine a-lactalbumin (1) and B. licheniformis b-lactamase (2)– and for mapping the area of interaction in an antigen-antibody model –as in hen egg white lysozyme (HEWL) and the monoclonal antibody IgG1D1.3 (3). The labeling phenomenon (methylation) is unspecific and depends primarily on the solvent accessibility of the polypeptide chain. This method addresses experimentally the measurement of the solvent accessible surface area (SASA) in biomolecules. So far 3H-DZN proved to be useful to accurately quantitate the extent of modification, even at very low levels (0.1-3% on a molar basis). However, this requires the handling of expensive radiotracers. In this context, we investigated the feasibility of a general labeling procedure based on non-radioactive methylene coupled to detection by mass spectrometry. Such analysis would permit to substantially reduce the needed amount of sample as well as potentially opening the possibility of automation. To achieve these goals one should increase the labeling yield. Here we present a device which takes advantage of the continuous dissolution and photolysis of DZN over the protein sample. The gas is generated into a 100ml glass syringe where the precursor salt (methylene diammonium sulfate) is mixed with an alkaline solution of sodium hypochlorite. An automated syringe drive unit controls gas flow (typically 0.6-1.0ml/min). DZN is injected through a stainless steel bubbler into a stirred protein solution (5mg/ml HEWL) placed in a capped quartz cuvette (1cm width x 1cm depth x 4.8cm height) thermostatted with air flow. An outlet tube is connected to a receiving syringe able to regulate the back pressure on the sample. The photolysis setup (Oriel) consists of a 1000W Hg/Xe arc source equipped with a back reflector, F/1 condenser lenses, IR water filter, automated shutter to control the time of exposure and a cut-off filter (l<300nm, Oriel 59044 code) to prevent damage to the protein chromophores. This arrangement allows the beam to be efficiently focused into the sample compartment, conveniently reducing photolysis lifetime. Thus, a steady-state concentration of DZN can be maintained by setting the in-flow rate and photonic flux on the sample. After dialysis and HPLC purification, HEWL samples were analyzed by electro spray MS to evaluate methylated products by pondering the distribution of mass peaks. 1) Craig P.O., Ureta D.B., Delfino J.M. (2002) Protein Science 11, 1353-1366 2) Daniela B Ureta, Patricio O Craig,  José M Delfino. (2001) Protein Science 10, suppl 1, 131. 3) G. E. Gómez, A. A. Cauerhff, P.O. Craig, F. A. Goldbaum, J. M. Delfino. (2003) Protein Science 12, suppl 1, 99.