INCORPORATION OF METAL IONS INTO POLYPHENYLQUINOXALINE

FeCl_3·6H_2O, NiCl_2, CuCl_2, ZnCl_2 and CrCl_3 have been incorporated into polyphenylquinoxaline by a new method. High-quality, flexible, glass-cast films have been obtained which exhibit increased glass transition temperature and excellent thermal stability. Moduli and tensile strengths of the metal-containing polyphenylquinoxaline films increase surprisingly at elevated temperature. Electrical resistivities of these films fall in the same order range as polyphenylquinoxaline alone. X-ray photoelectron spectroscopic study of metal-containing polyphcnylquinoxaiine films shows that all metals in these films are present in the ionic state, there is charge transfer between nitrogen of polyphenylquinoxaline and Cu~(2+), Zn~(2+) of CuCl_2, ZnCl_2 containing polyphenylquinoxaline films.     

INCORPORATION OF 11,12-DIHYDRORETINAL INTO THE RETINAE OF VITAMIN A DEPRIVED RATS

Abstract— The incorporation of 11,12-[15–³H]-dihydroretinal, a retinal in which the crucial 11-ene is saturated, into the retinae of vitamin A deficient rats as a result of intraperitoneal injection of the corresponding alcohol was shown by the presence of the tritium label in the rod outer segments and by identification of the extracted retinals using high pressure liquid chromatography. The amplitude of the electroretinogram (ERG) b-wave, diminished as the result of vitamin A deprivation, was not affected by administration of the analogue, although similar treatment of deprived litter mates with trans retinal restored the ERG b-wave amplitude to a normal level.
The evidence that the analogue is bound to opsin forming 11,12-dihydrorhodopsin is as follows: (1) when incubated with 11- cis retinal, extracts from vitamin A deficient rats regenerate 1.4 nmol rhodopsin while extracts from rats deficient in vitamin A and supplemented with 11,12-dihydroretinal regenerate 0.6 nmol rhodopsin indicating binding of the dihydroretinal blocks rhodopsin regeneration. (2) 11,12-dihydroretinal is shown to remain unchanged in hexane-washed retinae after extraction with methylene chloride and (3) injection of retinal into animals previously injected with 11,12-dihydroretinal also fails to restore visual sensitivity as measured by the ERG b-wave. Our results indicate that the dihydro-chromophore occupies the same binding site as the natural 11- cis retinal and that occupation of the chromophore binding site of opsin is not sufficient to restore the visual sensitivity in a vitamin-A-deprived animal.     

SAPO-34分子筛晶化过程中硅进入骨架的方式和机理

采用 Ｘ Ｒ Ｄ, Ｓ Ｅ Ｍ, Ｉ Ｒ 和 Ｎ Ｍ Ｒ 等手段考察了 Ｓ Ａ Ｐ Ｏ３４ 分子 筛的晶化过 程,深入研 究了晶化过程中硅进入 Ｓ Ａ Ｐ Ｏ３４ 晶格 骨架的方式和机理． 结果表明 ,在 Ｓ Ａ Ｐ Ｏ３４ 分子筛的整个晶化过程中没有 Ａｌ Ｐ Ｏ３４ 分 子筛晶相生成． 在 初始 凝 胶的 制备 过 程中, 模板 剂 的添 加和 混 合凝 胶的 老 化处 理对 Ｓ Ａ Ｐ Ｏ３４ 晶化过程的进行起着关键性的作用． 晶化前 期（ ＜ ２５ ｈ） , 硅原子直接参与晶核的形成和晶粒的长大过程,形成 Ｓｉ（４ Ａｌ） 结构,此阶段基本上 可以排除硅取代磷机理的作用; 晶化后期（ ＞ ２５ ｈ） ,少量硅以取代方式进入分子 筛骨架形成 Ｓｉ（ ｎ Ａｌ）（ ｎ ＝ ０ ～４） 多种硅结构     

INCORPORATION OF THE SPIN TRAP DMPO INTO CULTURED FETAL MOUSE LIVER CELLS

Abstract— The incorporation of four different spin traps into cultured fetal mouse liver cells was investigated using electron spin resonance spectroscopy. The cells were incubated in saline solutions of the traps, washed, and then irradiated in a saline solution containing hydrogen peroxide. The presence of a reproducible ESR signal was taken as evidence of spin trap incorporation. The spin trap DMPO was found to be incorporated, while PBN and 4-POBN were not. MNP was toxic to the cells.     

PHOTODESTRUCTION OF BACTERIORHODOPSIN

Abstract— Suspensions of purple membrane fragments showed obvious signs of degradation after illumination with intense pulses of light from 10 ns frequency doubled Nd: YAG laser at 532 nm with intensity densities in excess of 1 MW/cm². Using controlled illumination, a small fraction of the bacteriorhodopsin protein molecules were randomly destroyed in samples with a low salt concentration (12.5 m M ) and pH = 7.9. Calculations using information from the changes in the optical absorbance spectrum and transient changes in the optical absorbance spectrum during the photocycle support a model where one protein molecule of the bacteriorhodopsin trimer is photodestroyed, the other two protein molecules switch to a blue state . In the blue state , the protein molecules have a red shifted absorption, with a peak near 600 nm. The blue state molecules show transient absorption changes at 656 nm that are similar to the native bacteriorhodopsin, except the O state is missing or altered. Additionally, the changes in curvature of the purple membrane fragments that occur during the photocycle of intact protein molecules are severely depressed. The addition of salts to the photodestroyed suspension can change the blue state molecules back to a state with an absorption maximum at 568 nm. The salt ions probably shield the other members of the trimer from the photodestroyed protein. In these reconstituted samples, the O state is observed at 656 nm; however, the membrane bending is not observed.     

TIME-RESOLVED FLUOROMETRY OF BACTERIORHODOPSIN

Abstract A mode-locked Nd:YAG laser was used to excite the aromatic amino acid residues of bacteriorhodopsin in the purple membrane and the tryptophan (Trp) fluorescence decay analyzed with a streak camera (λ> 380 nm). The decay kinetics are resolvable into two first-order half-times (1.5 and 0.17 ns, respectively), while for retinylidene-free bacterioopsin, only the longer-lived Trp emission was observed. The shorter-lived species reappeared upon regeneration of bacteriorhodopsin by addition of retinal to bacterioopsin but not on treatment of the latter with an equivalent of retinol. It is proposed that these results are consistent with a structural model in which the 7-8 Trp's distributed among sections A, C, E and F of the seven helical segments A-G of native bacteriorhodopsin are distinguishable by their distances from the chromophore. Assuming a Förster mechanism for energy transfer with R_o= 25 and 32 Å, respectively, for retinylidene chromophore and retinol the Trp's may be divided into two groups: (i) those completely quenched by retinol and partly quenched by retinal (τ= 0.17) with R ≃ 18 Å and (ii) those (τ= 1.5 ns) which are quenched neither by chromophore nor retinol with R > ca. 30 Å. These results are consistent with and support some of the best models of Engelman et al. (1980) for the protein conformation in the purple membrane.     

PICOSECOND FLUORESCENCE SPECTROSCOPY ON INCORPORATION PROCESSES OF HEMATOPORPHYRIN DERIVATIVE INTO MALIGNANT TUMOR CELLS in vitro

Abstract— By using a highly sensitive streak-camera technique, we investigate incorporation processes of HpD into malignant tumor m-KSA cells in vitro. The picosecond decays of the total fluorescence spectra, the wavelength-resolved fluorescence decays and the time-resolved fluorescence spectra from HpD in the cells are measured as a function of the incubation time. The results show that the aggregate component of HpD which has a fast fluorescence lifetime of 100 ps and a red-shifted band of ∼ 660 nm selectively accumulates more and more in the cells with the increase of the incubation time.     

TS-1分子筛骨架钛原子引入过程的研究

采用廉价的四丙基溴化铵（ＴＰＡＢｒ）为模板剂合成了ＴＳ１分子筛．在晶化过程中,运用ＸＲＤ,ＩＣＰ,ＩＲ,２９ＳｉＭＡＳＮＭＲ和ＵＶＶｉｓ光谱等表征手段,系统地研究了钛原子引入分子筛骨架的机制,观察到钛原子随分子筛的形成同步进入骨架的规律．另外,尽管在晶化初期固相中没有ＴｉＯ２结晶出现,但存在分散态的ＴｉＯｘ物种．随晶化时间的延长,液相中钛物种之间聚合的几率增加,使固相中ＴｉＯ２晶体不断形成．     

THERMAL DECAY OF THE BATHOPRODUCT OF BACTERIORHODOPSIN

Abstract— The photoproduct of bacteriorhodopsin at 77 K is a stable, bathochromically shifted species, K. K begins decaying exclusively to the next bacteriorhodopsin photocycle intermediate, L, near 90 K. The thermal transformation from K to L is estimated to have a low barrier height with an upper limit of 3.8 kcal/mol.     

POSTREPLICATION REPAIR IN AN EXCISION-DEFECTIVE MUTANT OF ESCHERICHIA COLI: ULTRAVIOLET LIGHT-INDUCED INCORPORATION OF BROMODEOXYURIDINE INTO PARENTAL DNA*

Abstract— Ultraviolet (UV) light-induced incorporation of bromodeoxyuridine (BrdUrd) into parental DNA of an excision-defective mutant of Escherichia coli has been observed by selective photolysis of bromouracil (BrUra)-containing regions in the parental DNA. It appears that the BrUra-containing regions occur only in that DNA which has served as a template for normal semiconservative replication. After an exposure at 254 nm which results in one pyrimidine dimer per 45times 10⁶ daltons, incubation in BrdUrd resulted in BrUra–containing regions ˜ 1.5 times 10⁴ nucleotides in length at intervals of ˜ 55 times 10⁶ daltons in the parental DNA. Thus approximately one BrUra-containing region has occurred for every 1.2 pyrimidine dimers in the parental DNA. The observed incorporation of BrdUrd is interpreted in terms of a proposed model for postreplication repair in which genetic exchanges produce single-strand gaps in the parental DNA.     

THE EFFECT OF OXYGEN ON THE INCORPORATION OF MANGANESE,IRON AND COBALT INTO HEMATOPORPHYRIN IX IN GLACIAL ACETIC ACID

Abstract

The results of polarography studies for the reaction of hematoporphyrin IX with Mn(II), Fe(II) and Co(II) ion in acetic acid in the absence and the presence of oxygen are reported. The metal incorporation reaction in the presence of oxygen is quantitative for Mn and Co and incomplete for Fe. In the absence of oxygen, the Mn reaction does not proceed at all, whereas, both the Fe and Co reactions are quantitative.     

ULTRAVIOLET RESONANCE RAMAN SPECTROSCOPY OF BACTERIORHODOPSIN

Ultraviolet resonance Raman spectra of bacteriorhodopsin have been obtained using 229 nm excitation from a hydrogen-shifted neodymium yttrium aluminum garnet (Nd: YAG) laser. High signal-to-noise spectra are observed exhibiting vibrational bands at 762, 877, 1011, 1175, 1356, 1552 and 1617 cm-1 which are assigned to scattering from tryptophan and tyrosine side chains. This demonstrates the feasibility of using UV resonance Raman spectroscopy to monitor aromatic amino acid structural changes during the bacteriorhodopsin photocycle.     

RESONANCE RAMAN STUDIES OF BACTERIORHODOPSIN ANALOGUES

Abstract— We present the results of resonance Raman measurements on a series of bacteriorhodopsin (bR) analogues formed from synthetic retinals which have replaced the native chromophore in the active site. Specifically, 5,6-dihydro-bR, 13-desmethyl-bR, 10-methyl-bR, 14-methyl-bR, and 10.14-dimethyl-bR have been studied. All five analogues bind and form Schiff base retinal-apoprotein linkages. While the Schiff base linkages of 5,6-dihydro-bR, 13-desmethyl-bR, and 10-methyl-bR are protonated, like the native chromophore, the 14-methyl-bR, and 10,14-dimethyl-bR Schiff bases are unprotonated. These results suggest that the binding site of bacteriorhodopsin near the Schiff base moiety is different from that of rhodopsin. The protonated Schiff base -C=NH- stretching frequency of 5.6-dihydro-bR lies at 1660 cm^-1 which is unusually high for a bacteriorhodopsin based pigment. The downward shift upon deuteration is 16 cm^-1, essentially identical to that measured for bacteriorhodopsin. This and the other analogue results strongly reinforce our previous arguments that the Schiff base stretching frequency is determined in large part by two factors, the C=N force constant and the stretch interaction with C=N-H bend. On the other hand, the deuterium isotope effect is determined primarily by the stretch-bend interaction.     

COOPERATIVITY OF THE DEHYDRATION BLUE-SHIFT OF BACTERIORHODOPSIN*

Abstract– Dehydration of purple membrane (PM) causes a hlue-shift of the absorbance maximum from 570 nm to about 530 nm [Lazarev and Terpugov (1980) Biochim. Biophys. Acta 590 .324–338; Hildebrandt and Stockburger (1984) Biochemistry 23 ,5539–5548]. The absorbance spectra of PM dried in films at pH 0, 7 and 11 were measured at controlled relative humidities (RH). At pH 7, a blue-shift was observed similar to that previously reported. At pH 0(1M H₂SO₄) a reversible transition was observed from the “acid blue membrane” (maximum near 600 nm at 100% RH) to a blue-shifted dehydrated pigment (maximum near 578 nm at 50% RH), with isosbestic points at 592 and 710 nm. At pH 11 (NaOH) the absorbance maximum shifted to 530 nm, similar to the dehydrated form at pH 7. The fraction of hydrated chromophore, X_h, was calculated (assuming only two chromophore states, hydrated and dehydrated) as a function of humidity and pH. The resulting curve at pH 7 showed a steep decline in X_h below 20% RH. Near this hydration level, water clusters on protein surfaces break up, causing side-chain pK reversals. The Hill coefficient for the transition was about 2, indicating the minimum number of water molecules involved in a cooperative transition. The results suggest that as few as two water molecules are coordinated to the protonated retinal Schiff base of bacteriorhodopsin. A mechanism for the pH 7 dehydration blue-shift is proposed, involving a pK reversal of the protonated Schiff base and a nearby carboxyl side chain. At pH 0, a sharp decline in X_h occurs between 100 and 70% RH. Near this hydration level, complete protein surface coverage by a water monolayer occurs. The Hill coefficient is about 20, suggesting involvement of a large region of the surface.     

FLUORESCENCE OF BACTERIORHODOPSIN UNDER SUBPICOSECOND LIGHT EXCITATION

Abstract— A streak camera detection technique has been used to record the fluorescence of bacteriorhodopsin at room temperature induced by single subpicosecond light pulses. The fluorescence lifetime of bacteriorhodopsin has been found to be less than 2 ps.     

ANALYSIS OF PRIMARY PHOTOCHEMICAL PROCESSES IN BACTERIORHODOPSIN

Abstract— The sequence of primary events following light absorption by light adapted bacteriorhodopsin (bR₅₇₀) is considered by analyzing recent picosecond absorption and emission data. The analysis is facilitated by theoretical calculations which allow us to characterize the properties of the first excited singlet state. It is concluded that excitation leads to the eventual population of a photochemically important nonfluorescent excited state (I) which decays into a photoproduct (J₆₂₅)- In J₆₂₅, which is most probably a ground state molecule, the chromophore has undergone a structural change, presumably trans → 13- cis isomerization. It is suggested that the subsequent process

reflects a relaxation of the protein environment involving proton transfer.     

SPONTANEOUS AGGREGATION OF BACTERIORHODOPSIN IN BROWN MEMBRANE

Abstract— Halobacterium halobium cells grown in nicotine-containing medium synthesize bacterio-opsin (bO) but little bacteriorhod-opsin (bR) because nicotine inhibits retinal synthesis. In nicotine-grown cells, bacterio-opsin is found in a specific cell membrane fraction, the brown membrane (b.m.), which consists mainly of small round sheets. Freeze-fracture of isolated brown membrane reveals a dense particle population on its cytoplasmic fracture face, with few particles on the external face. There is no apparent order in the particle distribution and the fracture faces are practically indistinguishable from those of red membrane (r.m.). The absorbance spectrum of b.m. shows peaks at 550 and 410nm, the circular dichroism (CD) spectrum a positive band around 540nm and positive and negative bands around 410nm with a cross-over at 412nm. The photoreaction cycle of bR in b.m. has the same intermediates found in purple membrane (p.m.), but the apparent kinetics resemble those of bR monomers. Addition of 13-cis-, or all-trans-retinal to b.m. induces a rapid 5- to 10-fold increase in the visible absorbance band, and the absorbance maximum shifts to 560nm in the dark. Kinetic analysis of the absorbance increase shows three first order kinetic constants with t_½= 0.5 min, 6 min and 100 min, with most of the increase contributed by the fastest component. The CD bilobal pattern typical for purple membrane appears together with the strong negative band at 320nm. Ellipticity change at 590nm is nearly as rapid as the absorbance increase; within 5 min, 85% of the negative band is formed. Electron microscopy after addition of retinal reveals structurally distinct domains in the b.m. sheets which have the characteristic appearance of p.m. However, X-ray reflections are more diffuse compared to p.m. Flash spectroscopy of reconstituted b.m. detects the same photocycle intermediates as in b.m. or p.m., but with apparent kinetics closer to those of purple membrane than before reconstitution. These results show that bO in the b.m. binds retinal to form bR which spontaneously aggregates into a lattice. However, the reconstituted bR lattice is not as well-ordered as in p.m. and some bR is still present in the form of monomers and/or small aggregates.     

THE EFFECT OF CROSS-LINKING ON PHOTOCYCLING ACTIVITY OF BACTERIORHODOPSIN

Abstract— Purple membrane preparations from Halobacterium halobium were chemically modified with imidoesters. Dimethyl adipimidate (8.3 Å chain length) amidinates about five of the six free lysine residues whereas dimethyl suberimidate (11.3 Å) under the same conditions reacts with only 2–3 residues. Gel electrophoresis showed that the shorter chain length imidoesters were less effective than dimethyl suberimidate in oligomer formation. However, dimethyl adipimidate resulted in a more marked inhibition of the photoreaction activity. Monofunctional imidates, methyl acetimidate and methyl butyrimi-date, at comparable degrees of amidination, did not appreciably affect activity indicating that the presence of bulky groups on the exposed lysine residues does not cause the effects observed. Hence, the introduction of molecular mobility constraints by intramolecular cross-linking slows photocycling, and, therefore, inhibits proton pumping activity of bacteriorhodopsin. This indicates that conforma-tional changes of the protein moiety of bacteriorhodopsin occur during photocycling activity.