Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

Photocleavage of avidin by a new pyrenyl probe.

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py-biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py-biotin were demonstrated. Binding of Py-biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py-biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

High-sensitivity molecular recognition with light-induced polymerization.

A novel strategy for enhanced molecular recognition that utilized standard complexation strategies in combination with light-induced, or photo-, polymerization was recently demonstrated. Creative and rational materials design aided in the development of macroinitiators that produce a specific binding event, which is then amplified through photo-initation and chain polymerization. This polymerization-based amplification system produced a positive result that was visibly recognizable with amounts as low as 1000 molecules.

Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks.

Based on the enzymatic saccharification of the various pulps in the previous 0.8 l ultrasonic stirred tank reactor, the ultrasound-enhanced saccharification of waste papers such as newspaper, carton paper, office paper etc. was carried out in the same reactor as well as larger scale stirred tank reactors of size 3.2 and 6.4 l. The saccharification of each waste paper was less enhanced in the larger reactor at a given ultrasonic intensity. This could be attributed to the decrease in the ultrasonic intensity per reaction volume, i.e., the specific ultrasonic intensity. Most waste papers were more efficiently hydrolyzed with increasing specific ultrasonic intensities, although newspaper was less efficiently done for a too high specific intensity. Such an adverse effect might be due to the fact that some impurities in the newspaper such as lignin were activated by an intensive ultrasonic irradiation to form a rigid and closed network, which inhibited the access and adsorption of cellulase on to the substrate surface. The previous kinetic model was found to be applicable to analyze and simulate the saccharification of each waste paper in the different ultrasonic reactors. The ultimate conversion of a substrate based on the total sugar concentration estimated for an infinite reaction time could be correlated as a function of the ratio of initial substrate to enzyme concentrations at a fixed specific ultrasonic intensity. Either the apparent rate constant or the ultimate conversion increased and tended to approach a constant with an increase in the specific ultrasonic intensity except for the case of newspaper, while neither the apparent Michaelis constant, product inhibition constant nor glucose formation equilibrium constant was influenced by the specific ultrasonic intensity.

A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin.

A novel method is described to quantitate radiation-induced hydroxyl radicals in the vicinity of biomolecules in aqueous solutions. Coumarin-3-carboxylic acid (CCA) is a non-fluorescent molecule that, upon interaction with radiation in aqueous solution, produces fluorescent products. CCA was derivatized to its succinimidyl ester (SECCA) and coupled to free primary amines of albumin, avidin, histone-H1, polylysine, and an oligonucleotide. When SECCA-biomolecule conjugates were irradiated, the relationship between induced fluorescence and dose was linear in the dose range examined (0.01-10 Gy). The fluorescence excitation spectrum of irradiated SECCA-biomolecule conjugates was very similar to that of 7-hydroxy-SECCA-biomolecule conjugates, indicating the conversion of SECCA to 7-hydroxy-SECCA following irradiation. Control studies in environments that excluded certain radiation-induced water radicals for both the conjugated and unconjugated forms of irradiated SECCA demonstrated that: (1) the induction of fluorescence is mediated by the hydroxyl radical; (2) the presence of oxygen enhances induced fluorescence by a factor of about 1.4, and (3) other primary water radicals and secondary radicals caused by interaction of primary water radicals with biomolecules do not significantly influence the induced fluorescence. The data indicate that the induction of fluorescence on SECCA-biomolecule conjugates records specifically the presence of the hydroxyl radical in the immediate vicinity of the irradiated biomolecule. The method is rapid and sensitive, uses standard instrumentation, and the sample remains available for further studies.

Direct effects of radiation on the avidin-biotin system. Absence of energy transfer.

Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation. Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed. The loss of function of each compound was analyzed according to target theory. Target analysis revealed that the radiation-sensitive mass associated with the enzymatic activity and that associated with the fluorescence were unchanged by irradiation in the strongly coupled state. Therefore the noncovalent bonds between biotin and avidin do not permit the transfer of radiation-deposited energy in amounts sufficient to destroy the activity of apposing molecule.