Rapid freezing

Suspension samples will freeze well without any form of support, but during freeze-substitution they will disperse into the substitution medium. A dilute gelatin solution surrounding the sample will prevent this. 

Re-centrifuge and remove almost all the supernatant. Resuspend in gelatin. 

Introduce a small amount of the resulting suspension of sample plus gelatin into the nylon washer on the holder of the freezing device. The sample volume should be sufficient to form a meniscus above the level of the washer. 

Frozen samples can be stored under liquid nitrogen indefinitely. The method for freezing solid tissues is essentially the same as described in Protocol 8. Only the surface of the sample which contacts the freezing surfece will be well frozen, however, and even then only 10-15 jun of the sample will be free from ice crystal damage. It is important therefore that the surfece to be frozen is as flat as possible, not covered by a layer of fluid, and at the other extreme not allowed to dry. 

The acetone to be used for freeze-substitution must have the minimum water content possible. 1-2% water content will prevent substitution (11). 

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Knoll G 1995 Time resolved analysis of rapid events Rapid Freezing, Freeze-fracture and Deep Etching ed N Sievers and D Shotton (New York Wiley-Lyss) p 105... 

Jones G J 1984 On estimating freezing times during tissue rapid freezing J. Microsc. 136 349-60... 

Equipment for food freezing is designed to maximize the rate at which foods are cooled to —18° C to ensure as brief a time as possible in the temperature zone of maximum ice crystal formation (12,13). This rapid cooling favors the formation of small ice crystals which minimize the dismption of ceUs and may reduce the effects of solute concentration damage. Rapid freezing requires equipment that can deHver large temperature differences and/or high heat-transfer rates. 

Benzene, benzene-t/i, CFCI3 and CF3CCI3 were obtained commercially and were not further purified. Solutions of c a 0.3 - 1 volume % of benzene in CFCI3 were prepared in suprasil quartz tubes of 4 mm outer diameter on a vacuum line. The samples were degassed and sealed under vacuum (< 10-4 -porr). Polycrystalline samples were prepared by rapid freezing in liquid nitrogen. The samples were irradiated at 77 K for 5 minutes at an approximate dose rate of 250 G/min. using the radiation from an X-ray tube with a W anode operated at 70 kV and 20 mA. 

The mechanism of the first half-reaction has been studied by a combination of reductive titrations with CO and sodium dithionite and pre-steady-state kinetic studies by rapid freeze quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics 159). These combined studies have led to the following mechanism. The resting enzyme is assumed to have a metal-bound hydroxide nucleophile. Evidence for this species is based on the similarities between the pH dependence of the EPR spectrum of Cluster C and the for the for CO, deter-... 

ASTM C666-97 Standard test method for resistance of concrete to rapid freezing and thawing. ASTM Book of Standards Volume 04.02, 2001. 

As soon as the sample has been properly labeled and recorded, it should be placed in a generator-powered chest freezer located directly in the field. A flat-bed trailer can be used to transport freezers to and from the field site. Insulated boxes filled with dry-ice can be used as a substitute for freezers. However, chest freezers typically work better than dry-ice since they allow more cold air circulation around the samples, facilitating more rapid freezing. 

The time-dependent, rapid freeze-quench Mossbauer experiments with M. capsulatus (Bath) (51) indicate that decay of the peroxo species proceeds with the concomitant formation of another intermediate, named compound Q. This intermediate, observed in both the M. tri-chosporium OB3b (69, 70) and M. capsulatus (Bath) (51, 71) MMO systems by Mossbauer and optical spectroscopy, decays faster in the presence of substrates. Such behavior indicates that this intermediate is probably on the kinetic reaction pathway for hydroxylation (51, 70). 

Watson and Kenney [62] describe the use of high-performance size-exclusion chromatography to examine the aggregation of interferon-y and interleukin-2 after storage at elevated temperature, after mechanical agitation, and following rapid freeze-thaw. An excellent review on SEC can be found in Ref. 63. 

Air content of freshly mixed concrete by the pressure method Air content of freshly mixed concrete by the volumetric method Unit weight, yield, and air content of concrete Specific gravity, absorption, and voids in hardened concrete Resistance of concrete to rapid freezing and thawing Scaling resistance of concrete surfaces exposed to deicing chemicals... 

In the original rapid-freezing work on xanthine oxidase (53) it was found that in experiments employing about 1 mole of xanthine per mole of enzyme and an excess of oxygen, the time sequence of appearance of the various EPR signals was molybdenum (V), followed by flavin semi-quinone radical (FADH), followed by iron. This suggested that the electron transfer sequence might be ... 

The role of the iron-sulphur system of xanthine oxidase in the catalytic reaction is somewhat problematical. Nevertheless, it is clear, both from rapid freezing EPR (53) and from stopped-flow measurements monitored optically at 450 nm (58, 63) (where both iron and flavin are measured), that iron is reduced and oxidized at catalytically significant rates. Perhaps the best interpretation is that it functions as a store for reducing equivalents within the enzyme when this is acting as an oxidase, though it may well represent the main site of electron egress in dehydrogenase reactions (52). 

Fig. 5. Multiple phases in the reduction of xanthine oxidase by xanthine at pH 8.2. Intensities of the Rapid (circles) and Slow (triangles) molybdenum EPR signals expressed as electron/mole enzyme (i-e. per 2 atom Mo) are plotted as a function of time. Note the changes in the time scale. Rapid freezing was used for reaction times (at 22°) up to 1 sec. and manual mixing for longer times (at 25°) enzyme concentrations (immediately after mixing) were 0.09 mM and 0.13 mM respectively. The enzyme had Activity/A45o 125 corresponding to 63% of active enzyme and 20 mole xanthine/mole enzyme was used. (Data from ref. 67.)...

In conclusion, rapid-mixing/rapid-freezing EPR is a wonderful technique to obtain unique molecular structural information on biochemical reaction intermediates with high time resolution, but it is also experimentally sufficiently involved that one should either build up a dedicated lab with dedicated operators or turn to one of the existing groups that have the equipment and, especially, the developed skills to do these experiments. Be prepared to provide at least an order of magnitude more sample than required for a static EPR experiment. 

Another additional checklist to prepare for rapid freeze EPR experiments is as follows ... 

Try to imagine (e.g., from optically monitored stopped-flow experiments) what you can expect to get out of a rapid-freeze experiment, and preevaluate its biological relevance. 

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