Evaluation of Lyophilization Reagent Concentrate for the Preservation of Lactate Dehydrogenase from Mouse Tissue Lysates
The preservation of proteins, whether mixtures derived from lysates or purified, is done by a variety of widely used methods. The two primary methods of preservation, those being freezing and lyophilization, both rely on reducing water activity. Freezing aims to bind water in the form of ice while lyophilization (freeze drying) removes water. Free water is highly detrimental to the stability of many biomolecules and allows for enzymatic hydrolysis or chemical oxidation and hence its removal can lead to stability. However, both freezing and lyophilization of protein solutions are not necessarily routine procedures as in both processes the removal of free water can dehydrate proteins which leads to denaturation.
To simplify the freeze drying processes, a commercial additive (Lyophilization Reagent (2X) Concentrate, OPS Diagnostics) was used as an excipient for mouse tissue lysates. This reagent combines a lyoprotectant and matrix forming agent that are critical to forming stabile protein cakes. The reagent is a 2X concentrate so that it is mixed in equal volume with protein solution and then freeze dried.
Materials and Methods
Preparation of Tissue Lysates: Female CD1 mice, 42 days (Charles River Laboratories) were sacrificed and hind limb muscle, heart, liver, and kidney were extracted and homogenized with 10 volumes of PBS (mass to volume) with a conical glass homogenizer. The lysate was cleared by briefly centrifuging and the supernatant was transferred to clean tubes. An equal volume of Lyophilization Reagent (2X) Concentrate was added to each sample which were aliquoted (200 μl) into 1 ml screw cap glass vials. Half of the lysate samples were immediately frozen at -140°C while the other samples were freeze dried. The freeze drying cycle involved an initial freeze to ‑40°C, a primary drying temperature of -10°C for 16 hours, and a secondary drying phase of 20°C for 1 hour. Tubes were capped following the process and stored at 4°C until assayed.
Lactate Dehydrogenase Assay: Mouse tissue lysates from frozen and freeze dried samples were assayed for lactate dehydrogenase. Frozen tubes were thawed at room temperature while freeze dried samples were reconstituted with 200 μl water. A colorimetric NAD linked assay was used to measure released lactate dehydrogenase. The LDH substrate solution is prepared by mixing 2 ml of 1 M TRIS, pH 8, with 8 ml water, followed by the addition of 49 mg lithium lactate (Sigma L-1500), 100 μl of INT stock (33 mg/ml iodonitrotetrazolium chloride (Sigma I-8377) in DMSO), 0.9 mg phenazine methosulfate (Sigma P-9625), and 8.6 mg β-nicotinamide adenine dinucleotide (SigmaN-0632). Briefly, 50 μl of lysate (or lysate diluted in PBS for concentrated samples) is added to a well of a microtiter plate followed by 125 μl of substrate solution. The plate is placed in a kinetic plate reader and optical densities at 490 nm are measured every 30 sec. for 5 min.
Results and Discussion
Yields of lactate dehydrogenase from the four tissues were variable with muscle yielding the greatest and heart the least amounts of enzyme. The activity associated with either freezing or freeze drying was also variable with half the tissues showing higher activity with one process than the other (Figure 1). For muscle, kidney and liver, freeze dried LDH activity was 91, 111, and 87% that of the frozen samples. A significant difference was seen with the heart LDH as the lyophilized sample is 288% that of the frozen heart lysate. The explanation of this large activity difference can be attributed to either a freeze sensitive LDH isozyme produced by the heart or to lysate complexities generated during homogenization of the heart. In either case, heart LDH levels demonstrate that not all tissue lysates generate analogous enzyme activities when frozen and lyophilized.
Figure 1: Comparison of frozen and freeze dried lactate dehydrogenase activity from four mouse tissues lysates.
Freezing is the most popular method of preserving biomolecules and viable cells. However, cold storage of samples must be striated into different levels when discussing stability. Refrigeration, generally considered liquid samples kept around 4°C, may slow sample degradation, but it is generally only useful for very short periods. Freezing is the temperature range generally around -10 to -40°C and is useful for many liquid samples containing glycerol that prevents freezing. Freezing is not a good option for long-term storage of biomolecules or viable cells. Ultra low temperature storage is synonymous with -80°C freezers, a suitable environment for many nucleic acids and durable proteins and cells. However, samples at ultra low temperatures still have significant free water and will result in sample degradation over time. Cryogenic freezing, those freezers using high efficiency mechanical compressors or liquid nitrogen, can reach temperatures that bind all water and lead to true long term storage conditions. Consequently, when considering truly long term storage by freezing, cryogenic freezers are the only good option. Unfortunately, these freezers are expensive to both purchase and operate, but they do work well. Lyophilization, on-the-other-hand, requires relatively inexpensive equipment and has low operating costs. For the long term storage of proteins and microbes, freeze drying is a very viable option. It is cost effective and space efficient. Unfortunately it is not applicable to the storage of tissue culture cells.
Freeze drying and freezing are both effective methods used to preserve proteins. Depending upon the tissue source, LDH activity present in tissue lysates was comparable between frozen and freeze dried samples of lysate. The one notable exception was LDH from the heart which was more effectively preserved by freeze drying.