essential that in vitro esr observations be interpreted in terms of the chlorophyll species actually present. Chlorophyll is able to act both as electron donor and electron acceptor in chargetransfer complexes. The central Mg atom of chlorophyll is coordinatively unsaturated when it has the coordination number 4, and at least one of the Mg axial positions must always be occupied by an electron donor group. In the absence of other nucleophiles, the ketone C-O function in Ring V of one chlorophyll molecule serves as donor to the Mg atom of another, forming chlorophyll dimers, (Chl2), or oligomers, (ChlW)3. Extraneous nucleophiles (bases) can compete for the coordination site at Mg, with disruption of the chlorophyllchlorophyll interactions, to form chlorophyll-ligand adducts, (Chl-L). The nature of the nucleophile determines whether the chlorophyll-nucleophile adduct is monomeric or polymeric. Bifunctional ligands, such as dioxane, pyrazine, 1, diazobicyclo(2.2.2.)octane, and, in particular, water can cross-link chlorophyll molecules or chlorophyll dimers by coordination to Mg to form large (chlorophyll-nucleophile) micelles (to be published). The chlorophyll species present in a particular experiment are very sensitive to temperature, adventitious nucleophiles such as water, and solvent, factors not always taken into account in previous work. EXPERIMENTAL METHODSChlorophyll samples were dried, prior to solution preparation, by codistillation with CCL (36). The solvents used for in vitro measurements were first dried over Linde 3A molecular sieve and degassed under reduced pressure. Solutions were prepared and oxidant was added in a nitrogen-filled dry box or on the vacuum line.Irradiations were performed with a 150-W Varian Eimac lamp. Infrared components were removed by 2 cm of water and 2 dichroic infrared-rejecting filters. All irradiations were by red light with a Corning 2404 sharp cut-off filter. All in vitro measurements were made in 4-mm quartz esr tubes at -170'C; in vivo measurements were made at room temperature on concentrated slurries of cells held in a Varian water cell (V4548).625

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.

Thermal denaturation of protio-and deuteriophycocyanin, isolated from the blue-green algae Plectonema calothricoides, Phorrnidium luridum, and Synechococcus lividus, grown in H 2 0 and D?O, was studied in both H20 and DzO. The critical temperatures of thermal denaturation of deuteriophycocyanins were always significantly lower than those for protiophycocyanins, in both HzO and D20 solutions. The critical Institute of Applied Microbiology, University of Tokyo.1 The prefix "deuterio-" and the modifiers "fully deuterated" refer to phycocyanin containing 99.6% deuterium at all nonexchangeable positions. The prefix "protio-" refers to ordinary phycocyanin with hydrogen of mass 1 at all nonexchangeable positions.

The course of deuteriation was followed in mice given various concentrations of D2O in the drinking water. Equilibrium deuteriation in the body fluids is 95% attained by the 10th day, and from these values the experimental conditions leading to desired levels of deuteriation in mice are readily specified. The incorporation of deuterium into liver, kidney, and spleen reaches equilibrium by the 3rd week. Incorporation of deuterium into brain is much slower but eventually reaches approximately the same value. Median survival times for mice deuteriated at various D2O levels in the drinking water range from 60 days, for mice drinking 40% D2O, to 12 days for mice drinking 75% D2O.

The fully deuterated proteins C-phycocyanin, C-phycoerythrin, and cytochrome c have been obtained by biosynthesis with the leucine side chains, and only the leucine side chains, of normal ((1)H) isotopic composition. In these (isotopic hybrid) proteins, proton magnetic resonance analysis shows that the (1)H-leucine side chains are in a variety of environments. During protein biosynthesis, the alpha hydrogen of leucine is exchanged with a hydrogen ((2)H) from the aqueous medium.