Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

The dynamics of the enthalpy and volume changes found in the photodissociation of CO from sperm whale carboxymyoglobin and two site-directed mutants in which arginine-45 is replaced by glycine and asparagine are examined by photoacoustic calorimetry. An intermediate is observed whose lifetime at 20 degrees C is 700 ns. The enthalpy of the intermediate increases by approximately 7 kcal/mol upon replacing arginine-45 with either asparagine or glycine. These observations support recent proposals that an arginine-45 salt bridge is broken upon ligand dissociation.

The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellwsh Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (W) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or W. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either W or 488 nm. In vivo, GFP is excited by energy transfer from the Ca2+ -dependent blue light emission of the photoprotein aequorin ( 1 1, 20). A similar mechanism involving energy transfer from a photoprotein to a GFP has been demonstrated in a number of other bioluminescent coelenterates (5,(17)(18)(19). The covalently-boundp-hydroxybenzylidene-imidazolidinone chromophore responsible for the fluorescence of Aequorea GFP is derived from the cyclization of the amino acid residues Ser-dehydroTyr-Gly (4). This 0,-dependent cyclization reaction proceeds via a poorly understood mechanism which appears to be either autocatalytic, or at least catalyzed by ubiquitous enzymes, as hnctional GFP can be expressed in both prokaryotic and eukaryotic cells (3, 10). The GFP spectrum displays a major absorption peak at 395 nm and a minor peak at 470 nm. The fluorescence emission spectrum exhibits a major peak at 509 nm with a shoulder at 540 nm (18, 20,29).Although Aequoreu GFP has been identified and studied extensively since the late 1960s, recent isolation of the cDNA (22) and its subsequent expression in heterologous cell types (3, 10) has opened a new chapter for

Irradiation (350 nm) of air-saturated solutions of reagents containing an anthraquinone group linked to quaternary alkyl ammonium groups converts supercoiled DNA to circular and to linear DNA. Generation of linear DNA does not occur by accumulation of numerous single-strand cuts but by coincident-site double-strand cleavage of DNA. Irradiation forms the triplet state of the anthraquinone, which reacts either by hydrogen atom abstraction from a sugar of DNA or by electron transfer from a base of the DNA. Subsequent reactions result in chain scission. The quinone is apparently reformed after this sequence and reirradiation leads to double-strand cleavage.

Aequorea green fluorescent protein (GFP) has been expressed in a variety of cell lines and host organisms. A recent report (Heim et al.: Proc Natl Acad Sci USA 91:12501–12504, 1994) has documented that a GFP mutant with a single amino acid substitution (tyrosine 66 to histidinc; Y66H‐GFP) elicits altered spectral properties. Whereas wild‐type GFP emits with a maximum at approximately 509 nm (green fluorescence), Y66H‐GFP fluoresces with a maximum at approximately 448 nm (blue fluorescence). In this study we employed available argon and krypton ion laser lines to investigate the impact of laser excitation wavelength on the detection of Y66H‐GFP by flow cytometry. Using transiently transfected 293 cells, a cellular subpopulation with elevated blue fluorescence was detectable with excitation at 407 nm, but not with ultraviolet (UV), 458 nm, or 488 nm excitation. The blue‐fluorescing cells were further documented to express Y66H‐GFP by immunoblot analysis of sorted cells. Finally, we demonstrated the simultaneous analysis of both wild‐type and Y66H‐GFP in cotransfected 293 cells using 407 nm excitation while collecting blue fluorescence at 460 ± 20 nm (Y66H‐GFP) and green fluorescence at 525 ± 25 nm (wild‐type GFP). These studies illustrate the potential for assessing differential gene expression by simultaneously analyzing multiple GFP species with multiparameter flow cytometry. © 1996 Wiley‐Liss, Inc.