Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

Effect of α-amanitine on puffing and intranuclear RNA synthesis in Chironomus salivary glands

Incubation of Chironomus salivary glands with -amanitine in concentrations from 1 to 10 /ml results in the suppression of puffing and chromosomal ³H-uridine incorporation after 30 to 60 min in 80% of the cells. Nucleolar ³H-uridine incorporation remains completely unaffected. Even 4 h after the injection of high doses of -amanitine into living larvae, nucleolar incorporation is still pronounced. The distribution of resistant cells within the salivary glands suggests that the uptake of -amanitine is subject to physiological restrictions.—A puff typically induced during in vitro incubation of salivary glands was found to be less sensitive to -amanitine than the Balbiani rings.     

A study on relations of vegetation,climate and soils in Shanxi province,China

Relationships of vegetation, climate and soils in Shanxi plateau wereanalyzed by use of Canonical Correspondence Analysis (CCA). Shanxi province,located at 34°35–40°43 N, 110°15–114°33 E, was divided into a series of rectangular districts of30 latitude by 20 longitude. Areas of vegetation formations and soil types ineach district were measured carefully using fine grain on the vegetation andsoil maps of Shanxi. Climatic data were mean values of 25 years records in eachdistrict. Three data matrices of climate, vegetation and soil were combined byCCA. The results showed that the distribution of vegetation is closely relatedto the variety of climates and to soils distribution.     

Enhanced plant regeneration from microspore-derived embryos of Brassica napus by chilling,partial desiccation and age selection

Germination was readily induced in recalcitrant microspore-derived embryos of Brassica napus Topas when they were exposed to a period of chilling (9–12 days at 4°C) or partial desiccation (rapid or slow air drying) prior to germination. In general, embryos thirty-five days old had the highest germination rates as compared to younger or older ones. Populations of embryos were induced to germinate at a rate of over 90% under specific temperature, desiccation and age conditions. Comparisons to an embryogenic B. napus winter line, F346, are made.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

Adaptive Responses of the Larvae of Cirripede Barnacle Peltogasterella gracilis to Changes in Seawater Temperature and Salinity

The responses of the larvae of the cirripede barnacle Peltogasterella gracilis (Crustacea: Cirripedia: Rhizocephala) that parasitizes the hermit crab Pagurus pectinatus to different combinations of seawater temperature (25, 22, 20, 16, and 12°C) and salinity (from 34 to 8) were studied in a laboratory. The nauplii of P. gracilis completed the entire cycle of development at 22 to 12°C in a narrow range of salinity (from 34 to 28), which agrees well with the environmental conditions of the crab hosts' habitat. At favorable temperatures (22–20°C) and salinity (34–28), the nauplii reached the cypris stage in 88 ± 2 h, while at 12°C and 34–30, the naupliar development took 156 ± 5 h. The cypris larvae appeared more resistant compared with the nauplii, in terms of changes in both the temperature and salinity of seawater. They actively swam at all experimental temperatures and in the salinity range of 34–18. At temperatures (22–16°C) and salinities (34–24) favorable for the cyprids, their longevity in plankton equaled 6–10 days. Thus, the nauplii of P. gracilis is the more vulnerable stage of development in the life cycle of this parasitic barnacle. The tolerance against changes in environmental factors is due to the adaptive capabilities of parasitic larvae and the environmental conditions in the habitats of its host, a typical marine crustacean. The insignificant parasitization rate of the hermit crab by its rhizocephalan parasite may be explained by the death of the nauplii of P. gracilis, which occurs when they enter to the surface water layer.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Characterization of groups of the zygomycete genusRhizopus

Rhizopus is a zygomycetous genus. Several species of this taxon may infect humans and lower animals. Seventeen isolates ofRhizopus species in three distinct morphological groups were studied: the stolonifer group (sporangiophores greater than 1 mm in height, sporangial diameters of 100–275 µm, branched rhizoids); the arrhizus group (sporangiophores greater than 1 mm in height, branched rhizoids, sporangial diameters of 100–240 µm); and the microsporus group (sporangiophores less than 0.8 mm in height, sporangial diameters less than 100 µm, simple rhizoids). Maximal growth temperatures were characteristic: the stolonifer group grew at 30°C, the arrhizus group grew at 36°C, and the microsporus group grew at 45°C. The DNA mol% G + C base composition of all isolates ranged from 34.9 to 40.2% Species within the three groups were grouped by DNA differences. The arrhizus group was most distinctive with a value of 34.9–36.3%; the stolonifer and microsporus groups had G + C values of 37.0–39.3% and 37.8–40.2%, respectively. Our research clarifies and defines the G-C values of the three important groups ofRhizopus species.     

Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus

Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of ,-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40°C than at 80°C and was independent of the substrate concentration. The maximum yield of ,-trehalose from 10 mM maltose reached 86% at 40°C. In addition, ,-trehalose was also formed from maltose or ,-trehalose at 3.5% yield at 80°C. © Rapid Science Ltd. 1998     

Thermal stability and thermodynamic analysis of native and methoxypolyethylene glycol modified trypsin

Four methoxypolyethylene glycols (MPEG, molecular masses 350, 750, 2000 and 5000 Da), each activated by nitrophenyl chloroformate, were used to modify trypsin. Compared with the native trypsin, the MPEG-modified trypsin was more stable against temperature between 30°C and 70°C, longer chain of MPEG moiety corresponding to higher thermal stability. The T for the native and the modified trypsin (0.4 mg ml^–1) was increased from 47°C to 66°C. The stabilization effect caused by MPEG modification was the result of decreasing in both the autolysis rate and the thermal denaturation rate. The thermodynamic analysis of the thermal denaturation process showed that the activation free energy (G^*) of the native and the modified trypsin at 60°C was increased from 102.9 to 109.3 kJ mol^–1; the activation enthalpy (H^*) was increased from 57.4 to 86.9 kJ mol^–1; the activation entropy (S^*) was increased from –136 to –67 J molK^–1. A possible explanation for the decreased thermal denaturation rate caused by MPEG modification was also discussed.     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

Beiträge zur Züchtungsforschung an Pflaumen

Zusammenfassung 1. In den Wintern 1960/61, 1961/62 und 1962/63 wurde mit künstlicher Kälte (–13 bis –25 °C), die ein fahrbares Kälteaggregat erzeugte, die Frostresistenz von Pflaumenzuchtmaterial und einer Reihe von Pflaumenunterlagen im Freiland geprüft. Besondere Berücksichtigung fand dabei der natürliche Temperaturverlauf.2. Von den geprüften Pflaumengehölzen zeigte die geringste Frostresistenz eine Auslese von Prunus insititia und die Auslesen von Prunus cerasifera, besonders der tetraploide Klon B IV, 19,13, die diploiden Klone B IV, 20,6 und B IV, 17,15. Diese Typen erfroren nach einer zweitägigen, natürlichen Abhärtung im Dezember bei –20 °C; erst nach einer längeren Abhärtungszeit von 4 Tagen und mehr war ihre Frostresistenz gesteigert worden, so daß sie –20 °C schadlos überlebten. Nach kurzfristigen, witterungsbedingten Erwärmungen im Mittwinter wurden diese Formen derart schnell enthärtet, so daß sie bei Temperaturen um –15 °C erfroren. Nach längerem Tauwetter im Mittwinter erlangten sie nur sehr langsam eine erneute Frostresistenz.3. Insgesamt etwas besser zu beurteilen waren einige hexaploide Unterlagensorten, wie Ackermann, Brünker und Schwamborn 103. Sie tolerierten nach ztägiger Abhärtung –20 °C; Naundorf 102 und Brompton jedoch erst nach 4 Tagen. Ebenso stellt sich nach einer kurzfristig erfolgten Erwärmung nach ztägiger, erneuter Abhärtung wieder eine hohe Frostresistenz ein. Die relative Frostempfindlichkeit der geprüften Unterlagen kam darin zum Ausdruck, daß sie nach einer eintägigen Erwärmung im Mittwinter bei –24 °C total erfroren.4. Die beste Frostresistenz zeigten Vertreter der nordamerikanischen Pflaumen, wie Prunus americana und Assiniboine. Bereits nach einer zweitägigen Abkühlung Anfang Dezember überlebten sie schadlos –20 °C; nach 4 Tagen –25 °C. Ihre erreichte Frostresistenz war auch bedeutend stabiler als die der geprüften Auslesen von Prunus cerasifera und der hexaploiden Unterlagen.5. Es wurde der Versuch unternommen, einige der geprüften Pflaumentypen nach ihrer Frostverträglichkeit zu gruppieren, wobei die Merkmale Abhärtung und Enthärtung Berücksichtigung fanden.6. Zur Verbesserung der Frostresistenz der Pflaumen werden der Züchtung zwei Wege vorgeschlagen: 1. Herstellung von Nachkommenschaften aus genügend frostresistenten hexaploiden Formen; 2. Artbastardierungen mit bekannt frostresistenten Arten nordamerikanischer Herkunft.7. Neue Wege müssen auch in der Unterlagenzüchtung beschritten werden. Es wäre sinnvoll, dafür besonders diploide frostharte Arten als Kreuzungspartner zu berücksichtigen.

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Contributions to breeding research on plumsIV. Experiments on frost resistance of plum clones and rootstocks

Summary 1. During the winters of 1960/61, 1961/62 and 1962/63 a number of plum clones and stocks were tested outdoors for frost resistance. Artificial cold (–13 to –25 °C) was produced by means of a portable cooling system, taking natural temperature conditions into account.2. Of the plum woods tested, the lowest frost resistance was found in Prunus insititia and Prunus cerasifera, particularly in the tetraploid clone B IV, 19.13 and the diploid clones B IV, 20.6 and B IV, 17.15. These types froze in December after 2 days of hardening at –20 °C; only after 4 or more days of hardening was their frost resistance increased sufficiently to allow survival at –20 °C. After short warm spells in mid winter they became less hardy rapidly and froze at –15 °C. Prolonged thaw delayed their ability to acquire renewed frost resistance.3. The hexaploid plum rootstocks Ackermann, Brünker and Schwamborn 103 survived a temperature of –20 °C after a two day period of hardening; Naundorf 102 and Brompton required four days. A short warm spell followed by two days of hardening reinstated their high frost resistance, but the relatively high sensitivity of these plums to frost made them lose this resistance again after only one day's exposure to warm weather: they froze at 24 °C.4. Representatives of North American plum varieties such as Prunus americana and Assiniboine showed the highest resistance to frost. After two days of hardening they survived –20 °C undamaged, a temperature of –25 °C after four days. Their resistance to cold was more stable than that of Prunus cerasifera and of the hexaploid strains.5. An attempt was made to group the plums tested, using the characteristics hardening and loss of hardening as criteria.6. Two methods for improving frost resistance in plums are suggested to breeders: 1. The production of progeny from hexaploid forms that show adequate resistance to frost; 2. Interspecific hybridization with North American species known to be frost resistant.7. New means for the breeding of plum rootstocks must be used, and hardy diploid species are particularly suitable for this purpose.

     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

Whilst reliable estimates of chiasma frequencies can usually not be obtained, the probability (b) of a chromosome arm to be bound by at least one chiasma can often be determined. In the absence of interference this probability equals (1–e ^–2), where 2 is the average chiasma frequency of the chromosome arm and the average crossover frequency or map length. In the presence of interference is shown to retain its genetic meaning as an additive metric that may describe the chromosome arm or other distinctive chromosome segment in terms of genetic recombination. It is a form of potential map length, comparable to, but numerically different from the regular map length. It is termed provisionally crossing-over potential.A chromosome with armsm andn with crossing-over potentials and will form ring bivalents with a frequency (1–e ^–2).(1–e ^–2); open bivalents with a frequency (1–e ^–2).e ^–2+(1–e ^–2).e ^–2; univalent pairs with a frequencye ^–2.e ^–2. Estimates of these frequencies yield equations from which and may be solved. In rye (Secale cereale) their ratio (q) is approximately two and differs from the mitotic arm length ratio of 1.4, indicating localization of chiasmata in the long arms.Graphs are given to show how, with constantq, the relation between the probabilitiesb _m andb _n of the two arms being bound changes with changing averageb.Data are presented on chiasma frequencies in M I, and compared with the frequencies expected in the absence of interference to give an impression of the degree of interference. Apparent fusion of chiasmata simulates interference.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Influence of temperature on bionomics of cotton aphid, Aphis gossypii, on cotton

Life table parameters of Aphis gossypii Glover (Homoptera: Aphididae) on Gossypium hirsutum L. were determined at six temperatures (10, 15, 20, 25, 30, and 35 ± 0.5°C) in the laboratory. Relationships of life table parameters with temperature were described with mathematical equations. Development was fastest at 30°C, with a pre-larviposition period of 4.6 d. Survival to adult was greatest at 25°C (81%). Fecundity was highest at 25°C, with a total fecundity of 28.3 nymphs per female and a mean reproductive rate of 3.1 nymphs per female per day. Threshold temperatures for development in the first through fourth instar and the adult were 8.2, 8.0, 7.2, 6.2 and 7.9°C, respectively. The durations of these stages, expressed as temperature sums above these thresholds, were 24.2, 23.7, 23.0, 25.5 and 168.8 degree-days (D°), respectively. A. gossypii achieved its maximum net reproductive number (24.4 nymphs per female) and greatest intrinsic rate of increase (0.386 d–1) at 25°C. The high relative rate of population increase at 25°C results in a daily population increase of 47% and a doubling time of only 1.8 d, illustrating the tremendous growth capacity of A. gossypii populations under favourable conditions. Compared to literature sources, our source of A. gossypii, fed on cotton, showed a comparatively great heat tolerance.     

Residual motion of hemoglobin-bound spin labels and protein dynamics: viscosity dependence of the rotational correlation times

The residual motion of spin labels bound to cysteine 93 and to lysines of methemoglobin has been studied by electron paramagnetic resonance spectroscopy. To separate the influences of the solvent and the protein environment of the label fluctuations, the correlation times, , were analyzed as a function of temperature for fixed solvent viscosities, . Results show that over a wide range of viscosity the dependence of on may be empirically described by a power law ^k . The exponent k depends strongly on the location of the label on the protein surface. If one regards the spin labels as artificial amino acid side chains, characteristic values of correlation times and amplitudes of the rotational motion at the surface can be given. For =1 cP and T=297 K the correlation time of the labels bound to lysines is found to be =9 · 10^–10 s and the rotational diffusion is nearly isotropic. The spin label bound to cysteine 93 occupies a protein pocket, its rotational motion is therefore restricted. The correlation time of the label motion within a limited motion cone of semi angle =30° ± 3° is found to be =1.3 · 10^–9 s for =1 cP and T=297 K.