Henry L. Crespi

Proton Magnetic Resonance of Proteins Fully Deuterated except for 1H-Leucine Side Chains

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 (1H) isotopic composition. In these (isotopic hybrid) proteins, proton magnetic resonance analysis shows that the 1H-leucine side chains are in a variety of environments. During protein biosynthesis, the alpha hydrogen of leucine is exchanged with a hydrogen (2H) from the aqueous medium.

On the nature of the iron sulfur cluster in a deuterated algal ferredoxin

A protonated and a completely deuterated two-iron algal ferredoxin from Synechococcus lividus have been studied by optical, electron paramagnetic resonance, electron-nuclear double resonance, proton magnetic resonance and Mossbauer spectroscopies; temperature dependent magnetic susceptibility measurements are reported as well. These studies have confirmed the electron localized model of the active center in the two-iron ferredoxins, as previously deduced from studies of spinach ferredoxin, have yielded much more precise spectroscopic parameters for this center, and have thus greatly increased the confidence in this model.

Deuterium Isotope Effects in Cytology

Publisher Summary This chapter focuses on the growth characteristics of microorganisms and higher plants in deuteriated media and on the cytology of deuteriated cells. Deuteriated microorganisms exhibit a less well-organized pattern than do ordinary microorganisms. The degree of structural disorder is at a minimum in fully adapted cells, perhaps as a result of the successful confrontation of the kinetic disturbances encountered during adaptation. The kinetic problems of the adapting cells that lead to aberrant morphology can be minimized by serial subculture, or by fortification of the nutrient medium. Cultured animal cells are extremely sensitive to deuteriation. The differences in deuterium response, then, to a considerable extent are a reflection of the fundamental differences between plant and animal cells. Deuterium also affects cell division quite differently in plants and animals. Mammalian cells and sea urchin eggs are “frozen” in mitosis by what appears to be a markedly increased gelation of the cytoplasm, and the cells remain “frozen” as long as D 2 O is present. The chapter also discusses the cytochemistry of deuteriated cells, effects of deuterium on cell division, and some physical and chemical properties of D 2 O.

CULTIVATION OF MICROORGANISMS IN HEAVY WATER

Data are presented on the culture of a variety of algae and other microorganisms in highly deuterated systems. Growth rates for the unicellular green algae Scenedesmus obliquss and Chlorella ralgaris in 99.6% D/sub 2/O were improved so that they approach one-half the rates in H/sub 2/O nutrient media. S. obliquus was also successfully mass cultured in 5-liter units. The blue-green alga Plectonema calothricoides was grown on a continuous laboratory scale in 99.6% D/sub 2/O. A number of other green, blue-green, and red algae and diatoms have been cultured at D/sub 2/O levels from 75 to 99.6%. The bacterium Escherichia coli, the yeast Tonalopsis utilis, and the mold fungus Asperallus niger were grown in fully deuterated media. Protozoa have been cultured at somewhat lower D/sub 2/O concentrationsi Paramecium caudatum routinely in 60% D/ sub 2/O lettuce infusion and Paramecium bursaria in 50% lettuce infusion. In general, algae are best adapted to D/sub 2/O by serial subculyure, 0 to75 to 99 to 99.6% D/sub 2/O, but Chlorella, E. coli, T. utilis, and A. niger can be grown by direct transfer from H/sub 2/O to 99.6% D/sub 2/O nutrient media. (auth)

The growth of algae in deuterium oxide

Abstract Chlorella vulgaris, Chlorella pyrenoidosa and Scenedesmus obliquus can be grown in 99.6% deuterium oxide media. The rate of growth of the deuterated organisms is great enough to make it possible to use them as a practical source of biosynthesized fully-deuterated compounds.

The action of lysozyme on several blue-green algae.

Abstract The extraction of biliproteins from some varieties of blue-green algae is greatly facilitated by treating the algae with lysozyme (Berns, Crespi and Katz, 1962) . Fuhs (1958) previously had found lysozyme treatment to cause extensive disintegration of the filaments of Oscillatoria amoena , and envisaged the use of lysozyme to prepare algal protoplasts. In view of our work and Fuhs (1958) we have made systematic studies with lysozyme and have been successful in producing algal protoplasts.

Effects of Isotopic Substitution on Electron Spin Resonance Signals in Photosynthetic Organisms

Effects of Isotopic Substitution on Electron Spin Resonance Signals in Photosynthetic Organisms

Chloroplast pigments and photosynthesis in deuterated green algae

The isolation and characterization of deuteriochlorophylls a and b from the chloroplasts of fully deuterated green algae, together with some preliminary observations on photosynthesis in deuterated algae, are described. An improved procedure is given for the isolation and purification of the chlorophylls. The algae used in the study were cultured for more than a year in a nutrient solution prepared from 99.8% D/sub 2/0. The infrared studies reported provide independent evidence that the chlorophyll made by the algae is essentially free of hydrogen. The ratio of deuterio-chlorophyll a to b in crude extracts of the deuterated algae was determined to be in the range 1.5: 1 to 2: 1. The uptake of NaHC/sup 14/ O/sub 3/ by ordinary and deuterated C. vulgaris in H/sub 2/0 and D/sub 2/0 were measured, and the results are tabulated. (P.C.H.)

The chromophore-protein bonds in phycocyanin

Abstract Peptides formed by the proteolysis of C -phycocyanin were purified by TLC and analyzed for amino acid content. The data suggest that phycocyanobilin is linked to apophycocyanin through two bonds, one an ester bond involving the carboxyl group of an aspartic acid side-chain and the hydroxyl group of the enol form of ring A of the bilin, and the other a thio-ether type of bond from a cysteine side-chain to the bilin side-chain at position 2, a linkage analogous to that in cytochrome c. Liberation of phycocyanobilin from apophycocyanin by alcohols would involve transesterification at the ester bond followed by the formation of the keto form of ring A and elimination of the cysteine side-chain.

The location of retinal in the purple membrane profile by neutron diffraction

The trans-membrane location of retinal in the purple membrane of Halobacterium halobium, has been determined by low-angle neutron scattering studies on aqueous dispersions of the membranes. The membrane was bleached and regenerated with deuterated and with hydrogen-containing retinal. The modified retinal was obtained by extraction from bacteria grown in a totally deuterated medium. The determination of the retinal position is based on the differences in neutron scattering between a purple membrane sample with normal, protonated retinal and another sample with deuterated retinal. A distinct scattering density difference between the two preparations was observed. A direct structure determination was used with the retinal localized from a Fourier difference density profile. We conclude that the β-ionone ring portion of the retinal is situated centrally in the membrane.