Assessing the Value of Temperature Control on Centrifugal Vacuum Concentration

Introduction

Temperature effects are the nemesis of researchers doing protein purification. The heterogeneous nature of each protein species renders it with inherent characteristics that make potential temperature fluctuations a hazard to that protein’s stability. Indeed it is well know that high temperatures can cause denaturation, as well as freezing, but some proteins are even sensitive to low temperatures in that they are cold labile. Consequently when working with new biological extracts with the aim of protein purification, loss of activity may result from the traditional practice of chilling the sample.

A common technique used in protein purification is vacuum concentration. Gel filtration fractions and dilute extracts can be easily concentrated by the removal of water using products such as the Labconco CentriVap Centrifugal Concentrator. Samples are placed in the vacuum concentrator and centrifuged while a vacuum is applied. The centrifugation prevents the sample from splattering from tubes or microwell plates as a result of the pressure decrease. Under moderate vacuum, heating and cooling of the sample are also possible with the Labconco Refrigerated CentriVap. As noted below, temperature control can have an important impact on sample integrity.

Many samples which are vacuum concentrated contain inert or resilient solutes, as with inorganic ions or non-volatile organics. However, most biomolecules are susceptible to some type of degradation during processing. Major hazards are caused by proteases and nucleases which attack molecules in fresh lysates. Inhibitors can be used to suppress most enzymatic degradation, but a risk still exists that some of the desired biomolecules will be lost. Typically a combination of inhibitors and temperature control are used while manipulating samples. Using a temperature regulated centrifugal concentrator, such as the Refrigerated CentriVap, protection of biomolecules during vacuum concentration is also possible.

Effects of Temperature Control During Protein Purification

To illustrate the value of regulating sample temperature during protein purification, a liver tissue lysate was concentrated using a Refrigerated CentriVap which maintained the sample at 1°C (just above freezing) and with a Savant Speed Fuge operated at ambient temperature (22°C). A retained sample was stored at 4°C as an unprocessed control. Following concentration for one hour, distilled water was added to the samples to bring them up to their original volume. The samples were then assayed for lactate dehydrogenase activity (LDH) as a marker of protein stability.

LDH activity is measured spectrophotometrically at 490 nm using the INT assay. INT (i.e., Iodonitrotetrazolium chloride) is reduced by electrons stripped from lactate by LDH and transferred through NAD and the intermediate PMS (phenazine methosulfate). The reaction components are 1 part buffer (0.2 M Tris, pH 8), 1 part lactate (49 mM lithium lactate in water), 1 part INT/PMS/NAD, and 1 part enzyme solution. The INT/PMS/NAD solution is prepared before use by mixing 2.3 ml NAD (8.6 mg NAD [Sigma N-0632] in 2.3 ml water), INT (3.3 mg INT [Sigma I-8377] in 100 µl DMSO), and PMS (0.9 mg PMS [Sigma P-9625] in 100 µl water).

Tissue was prepared from mouse liver. Approximately 0.5 gm of liver was homogenized in a conical tissue grinder with 5 ml of phosphate buffered saline. The tissue lysate was centrifuged to clarify the solution which was further purified by passing the lysate over a PD-10 column (Sephadex G-25). The lysate was then divided into 0.5 ml aliquots in 1.5 ml microfuge tubes for vacuum concentration.

Samples were concentrated for 1 hr and then readjusted to 0.5 ml with water to standardize the samples. The samples were then assayed for LDH activity in a microplate reader.

After an hour of concentration, the sample processed at ambient temperature lost 35% of its activity as compared to the refrigerated vacuum concentrated sample. During this process, water loss from the ambient sample was faster due to the higher processing temperature, however proteolysis was also high. Reducing the sample to above freezing (1°C) resulted in slower water loss, but significantly higher LDH activity. Interestingly, the vacuum concentrated sample retained higher activity than the refrigerated control sample. This demonstrates the impact of slight temperatures differences on protein stability. The following graph summarizes this data.

Effects of Freeze/Thaw During Protein Purification

LDH is also recognized as an enzyme sensitive to freeze-thawing. The sensitivity of enzymes to repeated freezing and thawing of samples is well documented. However, some enzymes, such as LDH, can exhibit significant activity loss even after one freeze-thaw event. The significance of this problem as it relates to vacuum concentration is that aqueous solutions placed in high vacuums lose significant heat (molecular motion) to water as it changes phase from water to gas. Heat loss can be so significant that solutions can freeze, in affect resulting in lyophilization. Though freeze drying has many virtues, proteins that are frozen during concentration without chemical protectants (cryo or lyoprotectants) are often damaged. Furthermore, if the protein sample is subsequently thawed, i.e., concentrated but not to complete dryness, then the melting or collapse of the sample can further damage the solutes.

To demonstrate this concern, LDH samples prepared as noted above were concentrated in a CentriVap using a high efficiency rotary oil pump and a Refrigerated CentriVap equipped with a Welch DryFast diaphragm pump (Model 2042). During the vacuum concentration, samples in the CentriVap froze (due to the high efficiency pump) while those in the Refrigerated CentriVap remained liquid. After 1 hr of concentration, the samples were readjusted to their original volume and assayed as described previously.

The effect of freeze-thawing on LDH activity is significant (see chart below). The single freeze-thaw event which occurred during concentration resulted in an 85% loss of activity. Though this denaturation of the LDH could have been prevented by the addition of cryo- and/or lyoprotectants, the addition of such excipients is not necessarily desirable during purifications and concentrations of solutes.

Effects of Temperature on Evaporation Rate

Though controlling the temperature of sample during vacuum concentration has real and measurable value, it can also have a slowing effect on the concentration. The vaporization of solvent is accelerated as temperature increases, and inversely slowed by lowering temperature. With aqueous solutions, concentration is more effective at high temperatures and reduced as the samples cools. The following graph shows a comparison of water loss at 40°C, 20°C, and 4°C. This data illustrates that higher processing temperatures hasten the speed of water loss. Vacuum concentration at 4°C results in much reduced water loss, and thus requires longer processing times.

The data presented above leads to the obvious question of when is it necessary to use refrigerated vacuum concentration as it will require more processing time. The most obvious answer is when needed. The degree to which a protein sample is sensitive to degradation or denaturation should dictate the use of heat and refrigeration in processing. For known proteins, the parameters used in handling are defined by experience. However, when handling unknown proteins, lack of activity or poor yields may be related to sample handling issues as described above. Thus, for unknown (new) proteins, it may be prudent to concentrate using a device such as the Refrigerated CentriVap.

Summary

Protein samples can be adversely affected if vacuum concentrated at temperatures that allow proteolytic activity or result in denaturation. Both high and low temperatures can result in protein loss, and although inhibitors and protective agents can significantly reduce loss, regulating processing temperature can also serve to protect proteins. The Labconco Refrigerated CentriVap Vacuum Concentrator can supply and remove heat during sample concentration. Using LDH as a model protein, activity was preserved in protein samples by both decreasing proteolytic activity and prevent sample freezing.

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