Unfractionated preparation

To study the effect of veratryl alcohol, purified lignin peroxidase or unfractionated enzyme preparation was incubated with buffer, pH 3.0 or 5.0. The concentration of veratryl alcohol in the incubation mixture was 0, 10 or 100 mM. Incubation times were 38 days at 20°C and 40 days at 4°C. The protein concentration of purified enzyme was 80/tg/ml and of unfractionated preparation 180 tg/ml. The incubation mixtures were sterile filtered to prevent microbial growth. 

Effect of Veratiyl Alcohol. At pH 5.0 the purified lignin peroxidase was not inactivated under the conditions tested. At pH 3.0 the enzyme lost its activity when incubated at 20°C for 38 days, and the presence of veratryl alcohol could not stabilize it. 100 mM veratryl alcohol even inactivated the enzyme to some extent. Ionic strength did not significantly affect the activities. The effect of veratryl alcohol was the same when unfractionated enzyme was used. This time the ionic strength in the activity assay mixture affected the activities, probably because one enzyme in the unfractionated preparation is sensitive to high ionic strength 12),... 

Fig. 3 A and B. The weight-average length distribution of duplex AAV DNA molecules observed in the electron microscope has been plotted. A An unfractionated preparation. Circular molecules are shaded. B Fractions representing the major peak in a neutral sucrose gradient were observed. Reprinted by permission of J. Molec. Biol. 79 (1973)...

For preparative purposes batch fractionation is often employed. Although fractional crystallization may be included in a list of batch fractionation methods, we shall consider only those methods based on the phase separation of polymer solutions fractional precipitation and coacervate extraction. The general principles for these methods were presented in the last section. In this section we shall develop these ideas more fully with the objective of obtaining a more narrow distribution of molecular weights from a polydisperse system. Note that the final product of fractionation still contains a distribution of chain lengths however, the ratio M /M is smaller than for the unfractionated sample. 

For commercial grades of unfractionated PVP prepared by similar means (presumed to exhibit similar molecular weight distribution (MWD) and degree of branching), the following regression formula can be employed (71) ... 

The polymerization of 1,3,3-trimethyl-2,7-dioxabicyclo[2.2.1 Jheptane 35 was carried out in methylene chloride, toluene, and 1-nitropropane at temperatures between —78 and 0 °C32l Boron trifluoride etherate, triethyloxonium tetrafluoro-borate, antimony pentachloride, and iodine were used as initiators. Irrespective of the solvents and initiators employed, the products obtained at 0 °C were white powders with melting points of 50—55 °C, while those obtained at tower temperatures were sirups. The number average molecular weight of the unfractionated products ranged from 400 to 600. The molecular weight distribution of the oligomers prepared at 0 °C was broad, in contrast to the relatively narrow distribution of those obtained at -40 °C. 

Torres, A. R., Edberg, S.C., and Peterson, E. A., Preparative high-performance liquid chromatography of proteins on an anion exchanger using unfractionated carboxymethyl displacers, /. Chromatogr., 389, 177, 1987. 

Capture ELISA on selected oligomeric fractions of formalin-treated RNase A (see curves 3-7 in Fig. 15.9) also reveal that the plateau values increase with an increase in the number of cross-linked molecules in the fractions. This is due to an increasing proportion of bound epitopes per binding site or, in other words, epitope density on the surface. Thus, the nearly identical plateau values for the titration of native RNase A and formalin-treated unfractionated RNase A (curves 1 and 2 in Fig. 15.9) are fortuitous, being caused by the particular composition of oligomers present in the formalin-treated RNase A preparations that was analyzed. 

An interesting comparison is also given in Table XI86 of the action of malted barley alpha amylase upon corn starch, beta dextrins obtained from corn starch by the action of beta amylase, and upon corn amylose prepared according to the method of Meyer.6 90 These data show that, with equivalent amounts of enzyme, amylose was hydrolyzed much more rapidly and more extensively than unfractionated corn starch. Although the beta dextrins were hydrolyzed by this amylase, they were hydrolyzed much more slowly and less extensively than either of the other two substrates. These results are similar to those already considered for pancreatic amylase.41 64... 

If the expression of the mRNA results in a clear signal, a linear sucrose gradient (6-20% sucrose, 5 mM EDTA, 0.25% (w/v) sarcosyl, 15 mM PIPES-NaOH, pH 6.4) is prepared in SW27 tubes or equivalent and the mRNA is carefully loaded on top. After centrifugation (19 h, 80,000 xg), about 33 fractions of 1 ml each are collected, and the mRNA is precipitated using sodium acetate and ethanol. Prior to injection, the RNA of each fraction is pelleted, washed and dissolved in RNAse-free water as described above. Upon injection of 50 ng fractionated mRNA, a two- to fivefold increase in transport activity is expected for the positive mRNA-fraction compared with unfractionated mRNA. All other fractions should not show any specific functional activity. In cases where two neighbouring fractions induce transport activity, pooling of these should be considered. 

Low molecular weight lignin model compounds (synthetic phenyl-tetramers and Igepals ) were found to fit universal calibration. Fractions from preparative GPC, when analyzed by universal calibration, yield molecular weight distributions which add to a similar value to that found for the unfractionated parent sample. 

The anti-S. faecalis antiserum reacted with Lac-BSA amd the glycan as shown in the bottom panels of Figure 8. The amti-lac antibodies from this serum also reacted with these two immunogens yielding a similar pattern as the unfractionated amtiserum. It should be noted that the amtiserum amd amtibody preparations exhibited partial identity with these two antigens. The anti-gal antibodies also reacted with Lac-BSA amd the glycan but the precipitin bamds were quite different. 

Figure 6, illustrating some data from the analytical column, shows chromatograms of two 0.5% solutions of unfractionated BBB samples with [77] equal to 2.6 and 0.2 dl./gram Figure 7 shows chromatograms of three of the fractions of the unfractionated sample with [77] = 2.6 that were obtained from the preparative column. The product [77] M for these three fractions correlates satisfactorily with the function relating [77] M with Ve/Vy found for the polystyrene fraction. 

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