Concentration of dilute solutions of macromolecules by electrophoresis

An electrophoresis apparatus which is used for concentrating micrograms of macromolecules from solutions as large as 250 ml is described. The recoveries were greater than 91% with three different macromolecules tested (28 to 360 kDa). Solutions with volumes in the range of 35 ml were concentrated 70-fold in less than 90 min to a final volume of 0.5 ml. Larger volumes in the range of 250 ml were concentrated 227-fold in 16 h to a final volume of 1.1 ml. Sterile concentrates can be obtained if the apparatus is constructed under sterile conditions.     

A method for concentrating dilute solutions of macromolecules

A simple, inexpensive acrylate polymer which has a capacity to absorb 170 ml of water per g has been developed. It can be used to concentrate dilute solutions of macromolecules such as proteins, nucleic acids, and carbohydrates. The polymer absorbs only low-molecular-weight substances such as glucose, sucrose, and inorganic salts. It can replace the various conventional concentration methods. No special device or electricity is needed for the concentration. The inexpensive polymer, molded in the form of rods, can be very conveniently used as “disposable concentration sticks.”     

Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis the dependence of photoinhibition on fluence rate, duration and wavelength of irradiation were studied by measurements of oxygen production and fluorescence emission spectra. The analysis of the photosynthetic activity revealed that photoinhibition affects exclusively photosystem II (PS II), whereas photosystem I (PS I) remained largely unimpaired. Furthermore, PS II fluorescence emission decreased much faster in bleached than in unbleached controls.Studying the wavelength dependence of photoinhibition it was found that only radiation between 520 and 680 nm causes photoinhibition. This is about the same range of wavelengths which causes photobleaching. Fluorescence emission spectra of samples exposed to high fluence rates of 582 and 662 nm, respectively, essentially agree with those samples exposed to high fluence rates of white light, whereas the fluorescence emission spectra of samples exposed to blue light resemble those exposed to dim white light.NaN₃, a substance which prevents photobleaching, inhibits the photosynthetic O₂ production of Anabaena and, hence, enhances the photoinhibitory effect.     

Fluence rate and wavelength dependence of photobleaching in the cyanobacterium Anabaena variabilis

The fluence rate dependence of the photobleaching in the cyanobacterium Anabaena variabilis was studied under physiological conditions. According to the in-vivo absorption spectra measured every day during the 5 d exposition the phycobiliproteins are more sensitive to high fluence rates than chlorophyll a. The carotenoids are least sensitive, so that a relative, but not an absolute increase in the carotenoid content occurred. At very high fluence rates exceeding about 50 Wm^-2 white light the organisms were photokilled after 5 d of irradiation. Measurements of the nitrate concentrations during the experiments have shown that nitrate was not the limiting factor in these experiments. Analysis of the photobleaching kinetics at 13.5 Wm^-2 white light revealed that after about 8 d the contents of all the pigments studied have reached a new, constant level. After exposure of the photobleached cyanobacteria to low irradiances repigmentation occurred. Thus, photobleaching is a light adaptation process and not simply a photodamage phenomenon. Studying the wavelength dependence of photobleaching at a constant photon fluence rate of 4·10^-8 mol cm^-2 s^-1 we found that the photobleaching of both phycobiliproteins and chlorophyll a was exclusively caused by wavelengths absorbed by the phycobiliproteins, mainly phycoerythrocaynin, and red light absorbed by short wavelength chlorophyll. Wavelengths <520 nm were ineffective.     

Steady-state and transient electric birefringence of solutions of bent-rod macromolecules

We first calculate the steady-state birefringence, expressed in the form of specific Kerr constant, K_sp, of rigid, bent-rod macromolecules. Equations are derived for K_sp as a function of the geometric and electro-optical parameters. We also consider flexibly hinged rods and evaluate K_sp for them by averaging over the angle between the two arms, ?. Next, we turn to the time decay of the electric birefringence. The decay function for rigid bent rods is a sum of three exponential terms, and a procedure for their calculation is indicated. We observe that single-exponential decays can be found for ? > 90° or ? < 60°, in spite of the high electro-optical and hydrodynamic anisotropy of the macromolecule. Special attention is paid to the case of rods with equal arms.     

Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules

Dissolving biological macromolecules in dilute bicelle solutions, which form oriented liquid crystals in the presence of a magnetic field, permits measurement of anisotropic spin interactions such as dipolar couplings [Tjandra, N. and Bax, A., Science, 278, 1111–1114]. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. This paper demonstrates that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems. An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids.     

Volume effects in aqueous solutions of macromolecules containing non-polar groups

Specific volumes at 25° of aqueous solutions of poly(N-methyl acrylamide) and poly-(ethylene oxide) have been measured, and the data obtained is compared with corresponding data on low-molecular-weight compounds. The results support the notion, that the volume changes following the interaction between aliphatic groups and water are positive. They are discussed with particular reference to pressure effects on protein conformations.     

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.     

Free radicals from eumelanins: Quantum yields and wavelength dependence

Formation of light-induced free radicals from natural eumelanin (from bovine eyes) and synthetic melanin (from oxidation of 3,4-dihydroxyphenylalanine) has been studied by electron spin resonance spectroscopy. Action spectra measured for natural melanins are very similar to that found for synthetic melanin, and are unaffected by the removal of associated protein. A comparison of action spectra with optical absorbance spectra shows that the former has a more marked wavelength dependence, suggesting that the chromophore that is most active in free-radical production is not the major melanin chromophore that absorbs visible light. Measurements of quantum yields for freeradical production have been made over a wavelength range from 600 to 230 nm. The efficiency of radical production from natural eumelanin is about three times greater than from the synthetic material. Although production of the melanin radicals detected is independent of oxygen, some correlation with oxygen consumption is evident; quantum yields for radical production are approximately three times those for oxygen consumption obtained under similar conditions. Possible reasons for this are discussed.     

The wavelength dependence of some effects of ultraviolet radiation on in vitro DNA of phage alpha

The wavelength dependence of some of the effects of ultraviolet radiation on the physicochemical properties of DNA of phage α irradiated in vitro are discussed. An analytical ultracentrifuge and a spectrophotometer were used to study (a) the breaking of individual polynucleotide strands; (b) the local denaturation; (c) the presence of a fraction of molecules resistant to denaturation; and (d) the increase in the buoyant density of irradiated DNA. All the curves show a slight variation of the radiation efficiency in the range 2600-2800 A, and a well defined peak at λ = 2880 A.     

The temperature dependence of the surface tension of aqueous solutions of plasma proteins.

The surface behavior of aqueous solutions of fibrinogen, transferrin, gamma-globulin and albumin at the liquid-gas interface has been investigated by a modified Wilhelmy technique. The temperature dependence of the surface tension was studied over a temperature range of 20--80 degrees C and a pH range of 2--12. Most pronounced conformational changes of fibrinogen with this technique were found in physiological conditions: 35--45 degrees C and pH 7--8. A conformational change was found for gamma-globulin and transferrin solutions, but at a higher temperature and less pronounced than fibrinogen. Albumin did not undergo conformational transitions to a significant extent.     

The wavelength dependence of 8-methoxypsoralen photosensitization of radiation-enchanced reactivation in a mammalian cell-virus system.

The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus (Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet but not at others (240-289 nm). This same treatment induced RER in the near UV (302-370 nm) and the visible region (380-400 nm). An action spectrum for the photo-sensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.     

The turbidity maximum in the Tamar estuary

Many estuaries of medium to high tidal range exhibit an accumulation of fine cohesive material in their upper reaches in the region of the limit of saline intrusion. Much, or all, of this material is suspended each tidal cycle and the entire region undergoes a seasonal variation which appears to depend on fluvial input. Two factors which are throught to influence the formation and maintenance of turbidity maxima are the differing magnitudes of the bed shear stress (τ₀) on flood and ebb tides and the large vertical density gradient which developes on the ebb tide. Crucial to the importance of the first factor is that τ₀ exceeds a critical value, at which erosion occurs, for a greater period on the flood than on the ebb. The effect of the density gradient is that upward propagation of bed generated eddies is inhibited and the sediment is not transported into the upper part of the flow where it will be most effectively transported. It is not clear which, if either, of these mechanisms is dominant. Data consisting of vertical profiles of velocity, salinity and suspended solids were collected at four stations in the Tamar estuary during a high range tidal cycle. One station, at which the depth mean salinity (S _d ) varied from 0.0 to ∼ 12.0‰, was occupied permanently. The other stations were occupied such that data were collected asS _d varied in the range 0.0 to ∼ 4.0‰. In this way each station was occupied for a period of time on the ebb and flood tide. Observations show that during the early ebb, when the flow is relatively deep and slow, stratification persist untilS _d ∼ 0.0‰ and that no significant transport occurs while the flow is saline but that there is a rapid increase in suspended solids concentrations after this time. During the later ebb the shallower faster flow allowed the density gradient to be erode and significant transport was observed atS _d ∼ 5.0‰. On the flood tide the flow in the low salinity region is well-mixed troughout. Computation of the fluxes and total transport per unit breath of estuary show that on the ebb tide the quantity of solid material being transported by the low salinity (0–3‰) region remains nearly constant as this region of the flow is advebted seaward. On the flood tide, however, as the same region is advected landward the quantity of material being transported increases. It is concluded that in the Tamar estuary the early ebb tide stratification contributed to the formation and maintenance of a turbidity maximum which is strongly associated with the low salinity region of the flow. It is also speculated that the differences in the ebb and flood tide transport are caused by differences in the availability of mobile material on the bed at different stages of the tidal cycle.     

The cybernetics of biological macromolecules

This paper develops the concept of linkage as it applies to the binding, of ligands by a polyfunctional macromolecule, or system of macromolecules, under equilibrium, steady-state, and transient conditions.