Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

The effects of the buffer hepes on the division potential of WI-38 cells

Summary N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) and N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) have been found to be nontoxic substitutes for bicarbonate buffer in cell culture media. WI-38 embryonic human lung cells have been carried beyond the 60th passage with both HEPES and TES buffers.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Development of a buffer system for dialysis of bovine spermatozoa before freezing. I. Effect of zwitterion buffers

The effect of N-TRIS(hydroxymethyl)methyl-2-aminoetane sulfonic acid (TES); N,N BIS (2 hydroxvethyl)-2 aminoethane sulfonic acid (BES), N-2(hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES), morpholinopropane sulfonic acid (MOPS), and piperazine-N-N-BIS(2-ethane sulfonic acid (PIPES) solutions on dialyzed semen was studied. Each was titrated to pH 7.0 with TRIS-(hydroxymethyl)-amino methane (TRIS) solution and the osmotic pressure was adjusted to between 320 to 325 mOsm/kg. The new solutions were identified as TEST, BEST, HEPEST, MOPST and PIPEST, respectively. The solutions were used 1) alone, 2) in a composite with equal parts (V/V) of each solution and 3) in a 1:1 (V/V) combination with isosmotic trisodium citrate solution. Later, TRIS and sodium hydroxide (NaOH) were compared as titration bases for piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) and N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES). Ejaculates were diluted 1:10 (V/V) in extenders containing buffer, 20% egg yolk and 5% glycerol (V/V). The samples were dialyzed (1:50) during cooling for a period of 2 h. Each sample was dialyzed against the same buffer system containing 5% glycerol without egg yolk and later it was frozen in pellets. The treatments were evaluated by observation of sperm motility in fresh and thawed semen samples. The latter were also analyzed by electronic count of cells that passed through the Sephadex column. Sperm survival was higher in PIPEST (PIPES titrated with TRIS) or the composite buffer, and the inclusion of 50% sodium citrate (Na citrate) improved significantly (P<0.05) sperm motility in fresh and frozen-thawed semen samples. There was no difference (P>0.05) between the titration bases. In the second experiment, sperm survival was superior in extenders containing PIPEST (P<0.05) than in those containing TEST independently of the inclusion of Na citrate.     

Acid growth effects in maize roots: Evidence for a link between auxin-economy and proton extrusion in the control of root growth

The role of proton excretion in the growth of apical segments of maize roots has been examined. Growth is stimulated by acidic buffers and inhibited by neutral buffers. Organic buffers such as 2[N-morpholino] ethane sulphonic acid (MES) — 2-amino-2-(hydroxymethyl)propane-1,3 diol (Tris) are more effective than phosphate buffers in inhibiting growth. Fusicoccin(FC)-induced growth is also inhibited by neutral buffers. The antiauxins 4-chlorophenoxyisobutyric acid (PCIB) and 2-(naphthylmethylthio) propionic acid (NMSP) promote growth and H⁺-excretion over short time periods; this growth is also inhibited by neutral buffers. We conclude that growth of maize roots requires proton extrusion and that regulation of root growth by indol-3yl-acetic acid (IAA) may be mediated by control of this proton extrusion.Abbreviations IAA indol-3yl-acetic acid - ABA abscisic acid - FC fusicoccin - PCIB 4-chlorophenoxy-isobutyric acid - MES 2(N-morpholino)ethane sulphonic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3-diol - NMSP 2-(naphthylmethylthio)propionic acid     

Effect of N-tris (Hydroxymethyl) Methylglycine and N-2-Hydroxyethyl-piperazine-N'-2-Ethanesulfonic Acid Buffers on Linolenic Acid as a Substrate for Flaxseed Lipoxidase

The delay in, or loss of, flaxseed lipoxidase activity in N-tris (hydroxymethyl) methylglycine and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffers with linolenic acid as a substrate appears due to an alteration of the lipid micelle. Flaxseed lipoxidase activity is dependent on the ionic strength of the assay solution. These effects are not observed with linoleic acid as substrate. The influence of these 2 buffers on linolenic acid micelles may have a direct bearing on recent reports of chloroplast structure and activity in these buffers.     

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.     

Acid dissociation constants and pH values for standard "bes" and "tricine" buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and -25 degrees C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H2O mixed solvents at subzero temperatures from -20 to 0 degrees C, with the intention of establishing a pH (designated pH*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid ("bes") and N-tris(hydroxymethyl)methylglycine ("tricine"), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H2(g,l atm) /Bes, Na Besate, NaCl / AgCl;Ag and Pt;H2(g,l atm) /Tricine, Na Tricinate, NaCl /AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Buffer)+/- in equilibrium with(Buffer)- + H+.     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

The effect of different buffers on terminal deoxynucleotidyl transferase activity.

The polymerization of dATP, dCTP, and dGTP onto the defined length initiator, d(pA)10, has been carried out in four buffers. The relative effectiveness of the buffers for the polymerization of each deoxynucleoside triphosphate decreased in the order: cacodylate, 2(N-morpholino)ethane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and Tris. With the poorer buffers, activity could be increased by the addition of KCl: this effect is not primarily due to an increase in ionic strength. With dGTP as the substrate, but not with dATP or dCTP, activity increased when the concentration of the more active buffers was raised beyond 0.2 M.     

锰——超氧化物歧化酶活力测定的五种方法比较研究

 本文分别以CN~-抑制和SDS处理区分Mn-SOD与CuZn-SOD,对五种SOD活力测定方法进行了比较研究。结果表明:(1)化学发光法和光化学扩增法不适用于Mn-SOD活力测定,CN~-和SDS对这两种方法有明显的干扰作用。(2)NBT还原、Cyt c还原和亚硝酸盐形成法都能用于Mn-SOD活力测定,用这三种方法测得Mn-SOD每活力单位相当于酶的含量分别为2.93μg、0.11μg和0.028μg。说明NBT还原法灵敏度最低,其次是Cyt c还原法。亚硝酸盐形成法灵敏度高,专一性强,为五种测定方法之首。     

Acetic acid from H2 and CO2

Cell extracts of a nonsporeforming strictly anaerobic bacterium, Acetobacterium woodii produced acetate in N-tris(Hydroxymethyl)methyl-2-aminoethane sulfonic acid or phosphate buffers from hydrogen and carbon dioxide. The formation of acetate was not dependent on the presence of ATP in the reaction mixture; ADP also did not influence the acetate production. Since acetic acid is the main fermentation product during growth of A. woodii with H₂ and CO₂, ATP must be synthesized in the course of acetate formation. The possible sites of ATP synthesis are discussed.     

Use of amine buffers for in vitro studies of bone resorption

Summary Responses of cultured fetal rat bones to parathyroid hormone (PTH) were compared in media gassed with 5% CO₂ and buffered with bicarbonate or in media buffered with the amine buffers HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), Tris (tris(hydroxymethyl)aminomethane), or glycylglycine. PTH-induced bone resorption was markedly reduced in media buffered with the amine buffers and cultured in air in the absence of added bicarbonate. When the amine buffers were added to bicarbonate-buffered media, they decreased the responses of the bones to submaximal concentrations of PTH. But the response to supramaximal concentrations of PTH was usually not impaired. The results confirm our earlier studies indicating a CO₂ and/or bicarbonate requirement for optimal bone resorption. They also demonstrate that the amine buffers can inhibit the responses of bone to PTH even in the presence of CO₂ and bicarbonate. Supported by NIH research Grant #AM 11262. Supported by NIH Research Career Development Award #AM 70210.     

Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum.

The photosynthetic oxygen evolution capacity of Agmenelium quadruplication suspended in four hydrogen ion buffers (pH 7.4, 0.05 M) and its synthetic marine growth medium was measured with an oxygen electrode. High rates of oxygen evolution were obtained in the growth medium and N-tris(hydroxymethyl)-methylglycine (Tricine) buffer. Compared to oxygen evolution in the growth medium, rates in phosphate buffer and N-tris(hydroxymethyl)-2-aminoethanesulphonic acid (TES) buffer were sometimes reduced by up to 30% and rates in tris (hydroxymethyl) amino-methane (Tris) were consistently reduced by 50%. An incubation-rinsing procedure caused inhibition of oxygen evolution in TES, phosphate, and Tris by 50 to 100%. Oxygen evolution could be restored to cells rinsed in TES or phosphate by resuspension in growth medium or in buffer plus magnesium and calcium ions. Bezoquinone-supported oxygen evolution was not affected by rinsing with any buffer tested except Tris. Ferricyanide was photoreduced at a low rate by cells rinsed in Tes but at a high rate in TES plus magnesium and calcium ions. We interpreted our results to mean that, in Agmenellum quadruplicatum, inhibition of photosynthetic oxygen evolution by Tris occurs at the level of photosystem 2 while the effects of TES and phosphate are on electron-transport occurring after the rate-limiting reaction.     

Buffer-dependent variations in assay of tyrosine aminotransferase holoenzyme

Homogenization of rat liver in Hepes (N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid), MOPS (2-[N-morpholino]ethanesulfonic acid), Na phosphate, Pipes (piperazine-N,N′-bis[2-ethanesulfonic acid]), TEA (triethanolamine), TES (N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid), Tricine (N-tris-[hydroxymethyl]methylglycine), or Tris (tris[hydroxymethyl]aminomethane), and subsequent assay for supernatant total and holo tyrosine aminotransferase activity using these buffers yields apparent enzyme concentrations which vary depending upon the buffer composition, the ionic strength, and the fold-dilution of the supernatant. A precipitous decrease in the apparent holoenzyme concentration results from a slight dilution of the supernatant with most of the buffers. Some of the dilution effects may be due to dissociation of pyridoxal phosphate from the apoenzyme or to competition between the buffer and pyridoxal phosphate for association with the enzyme. The percentage of the apparent total enzyme which exists as holoenzyme varies from 3% for supernatant prepared in Na phosphate buffer up to 94% for that prepared in Hepes. Inactivation of total enzyme activity occurs to a similar extent resulting from incubation of liver homogenates prepared with Na phosphate, Hepes, or Pipes. The residual apparent holoenzyme activity observed when assayed in the presence of Na phosphate may be due to reaction of an enzyme other than tyrosine aminotransferase. The data provide a basis for explaining the large variation in reported percentage holoenzyme and should also serve as a warning for other holoenzyme assays which use pyridoxal phosphate as a cofactor.     

Production of Interferon in Serum-Free Human Leukocyte Suspensions

The recovery of interferon from Sendai-infected suspensions of purified human leukocytes is dependent on the serum concentration in the incubation medium. Very little interferon is obtained from serum-free suspensions. The data reported demonstrate that the critical macromolecular, age-independent, and species-unspecific serum principle can be omitted from the suspensions if the medium is supplemented with a combination of crystalline serum albumin and high concentrations of any one of five studied dipolar ionic buffers [N,N-bis(2-hydroxyethyl) glycine (Bicine), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (TES), and N-tris(hydroxymethyl)methylglycine (Tricine)]. The optimal combination [TES (1.0%, w/v) and bovine serum albumin (0.8%, w/v)] allows the production of potent preparations of serum-free human interferon.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. I. Analysis of surface-localized proteins

Lactoperoxidase-catalyzed iodination of chloroplast membranes has been employed to characterize the vectorial distribution of lamellar proteins. The enzymatic reaction is highly specific for only the outermost membrane components (Phillips, D. R. and Morrison, M. (1971) Biochemistry 10, 1766–1771); we have determined the distribution of ¹²⁵I label and changes in photochemical activities after iodination in an effort to identify these components. Three major conclusions are evident:

1. 1. The coupling factor for photophosphorylation is highly exposed and is selectively and rapidly inhibited by the iodination reaction.

2. 2. A loss of Photosystem I activity (NADP reduction) resulted from iodination. Partial reactions indicated the effect was on electron-transport components on the reducing side of Photosystem I. There was also a limited inhibition of methyl viologen reduction.

3. 3. Iodination of intact membranes caused a reduction in rates of Photosystem II-dependent Hill reaction activity. This inhibition could not be explained solely on the basis of iodination effects on electron-transport components involved in the oxidation of water. The implications of these data with respect to previous chloroplast-membrane models are discussed.

Interactions of bovine serum albumin with biological buffers,TES, TAPS,and TAPSO in aqueous solutions

The thermal stability of bovine serum albumin (BSA) in the aqueous solutions containing the biological buffers, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), N-[tris(hydroxylmethyl)methyl]-3-aminopropanesulfonic acid (TAPS), and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), was studied by using dynamic light scattering (DLS) at various temperatures and concentration ranges of buffers. It is found that the increase of the buffer concentration enhanced the thermal stability of protein BSA, and the stabilization tendency follows the order of TAPSO > TES ≈ TAPS. In this study, we have also investigated the interactions of BSA with TES, TAPS, and TAPSO by using various techniques, such as UV–vis absorption, fluorescence, and molecular docking. It is revealed that the main interactions between the studied buffers and the peptide moieties of proteins are electrostatic including hydrogen bonds. The results obtained from this series of studies confirmed that the biological buffers, TES, TAPS, and TAPSO can serve as good stabilizers for the globular protein BSA, in the aqueous solutions.     

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.