Itinerario Formativo, testimonio de vida y misión

For years, research laboratories routinely stored enzymes in frozen solutions because it is believed that the long-term stability of enzymes in frozen solution is greater than in unfrozen solutions. However, in some cases, freezing and thawing may cause substantial enzyme denaturation, which can be detected as alterations of the enzyme structure or loss of catalytic activity. The low temperature perse and pH-shift of the solution as it cools (85, 86) usually viewed as the main causes of enzyme denaturation in frozen storage. For this reason, freezing can induce reversible inactivation of pH-sensitive enzymes.

4.1. pH-shift in storage buffers. Although buffers are often used to store enzymes in

order to maintain constant pH, evidences for pH shift upon freezing due to precipitation of the less-soluble buffer component have been well documented. For Tris, citrate, and potassium phosphate, the pH has been reported to change up to +1, -0.4, and +0.6 unit, respectively, upon freezing (87, 88). The most dramatic pH changes have been observed with disodium phosphate (Na2HPO4.12H2O) (85, 86) up to 3.5 pH units. In work by Pikal-Cleland et al., protein

2 _{With the crystal structure of the nitroalkane oxidase obtained just recently by Nagpal et al.(80),}

the tyrosine that has been identified in the study by Gadda et al. (79) was proposed to be at position 398.

denaturation during freezing and thawing in potassium phosphate (KP) and sodium phosphate (NaP) buffer was studied (86). Tetrameric β-galactosidase was chosen in this study because its inactivation can be monitored with large structural changes from tetramer to monomer. In addition, earlier studies indicated that this protein lost activity when its subunits dissociated at pH ≤ 6 (89, 90); the pH optimum for protein stability is between 6 and 8 (91, 92). In that study, the pH changes during freezing were determined under nonequilibrium conditions. A combined pH electrode was used with a reference solution containing potassium chloride, glycerol, and formaldehyde that kept the electrolyte solution from freezing and enabled pH measurements down to -30 oC. When the enzyme was stored at -10 oC in 100 mM or 10 mM NaP with initial pH of 7.0, the pH significantly decreased to 3.8 or 5.5, respectively. In contrast, when the enzyme was stored in 100 mM or 10 mM KP at -10 oC and pH 7, the pH of the frozen solutions were 7.3 and 7.1, respectively. The magnitude of pH shifting in both buffers was dependent on buffer concentrations with higher concentrations giving salt crystallization upon freezing. In the ice-liquid interphase, water formed ice crystals and escaped from the buffer phase, resulting in a more solute-concentrated buffer. The precipitation of buffer salts can happen at different times and at various temperatures, depending on their solubility and buffer concentrations (93, 94). Ice crystals usually form before the precipitation of buffer salts. These crystals pull the basic phosphate species (i. e., HPO42-) away from the solution, thereby inducing a pH shift due to the

change in the ratio of base to acid in the buffer. In the case with NaP and KP buffers, disodium salt is much less soluble in water than potassium salt because their eutectic points are -0.5 oC and -13.7 o_{C, respectively. Therefore, the HPO}

42- species of NaP buffer was depleted from solution

much earlier than that of KP buffer leading to a greater change in pH. For β-galactosidase, the lower recovery of activity and larger structural alterations was observed after freezing and

thawing in NaP buffer than in KP buffer. This was used as evidence for the larger pH shift of NaP. The effects of the freezing and thawing rates on activity recovery of the enzyme were also investigated using different cooling and warming methods. Data on activity recovery and infrared spectroscopy of the enzyme structure revealed that less enzyme was denatured when fast cooling and fast warming methods were used. This implied that rapid freezing and thawing can minimize the time of exposure of the enzyme to low pH and high salt concentrations, thereby resulting in less denaturation of enzyme from storage.

4.2. Reversible inactivation of enzymes. In limited cases, freezing can induce reversible

inactivation of pH-sensitive enzymes; this has been observed in the case of L-amino acid oxidase (LAAO) from Crotalus adamanteus (95). LAAO is a snake venom enzyme that catalyzes a flavin-dependent reaction involving the oxidative deamination of a number of L-amino acids. In that study, the enzyme was stored in 0.2 M Tris-HCl, pH 7.8, and frozen at temperatures between -5 to -60 oC. The maximal inactivation was observed when the enzyme was stored at -20 oC, as suggested by the dramatic loss of enzymatic activity. Repeated freezing and thawing did not affect the rate of inactivation. No inactivation was observed in the absence of freezing. The degree of inactivation was directly dependent on Tris-HCl buffer concentrations between 2 mM to 200 mM, but was independent of enzyme concentrations in storage. Under anaerobic conditions, a protective effect against freezing inactivation was observed in the presence of substrates, such as L-leucine and L-arginine. However, there was no protection when the enzyme was reduced anaerobically with dithionite. Glycerol and dimethyl sulfoxide also protected the enzyme. This was explained with the hypothesis that glycerol and methylsulfoxide influenced the ice structure in a way that prevented the formation of the inactive form of the enzyme. Spectroscopic data from protein fluorescence showed that the inactive enzyme

maintained its native conformation. UV-visible absorbance spectra of the enzyme-bound FAD cofactor showed the inactivation of the enzyme was associated with 7 nm and 12 nm hypsochromic shifts of the 462 nm and 390 nm peaks, respectively. Photoreduction in the presence of EDTA was used to examine the reduction properties of the FAD in the inactive enzyme. The rate of the reduction was 10-fold lower than that of active enzyme and only 50% of flavin semiquinone could be formed. Based on spectroscopic and photoreduction data, the authors concluded that the microenvironment of the flavin in the inactive form of the enzyme was significantly affected. The reactivation process of LAAO was also investigated. The enzyme was fully reactivated at pH 5 by increasing the temperature. Adjustment of the pH to 5 or an increase in temperature alone could not reactivate the enzyme; both were required for a full reactivation of LAAO. When the UV-visible absorbance of FAD was monitored during the reactivation process, the absorbance spectrum eventually became similar to that of the native enzyme, which further supported the conclusion that the inactivation of the enzyme was associated with changes in the flavin microenvironment. The temperature-dependence of reactivation yielded a ∆H‡ of 36 to 41 kcal mol-1 (151 to 171 kJ mol-1). These large ∆H‡ values from temperature reactivation were in conflict with spectroscopic data, which showed no large protein conformational change. The authors explained the effect of temperature on freezing inactivation using the following model:

E_act E* k E_in

where Eact and Ein represent the active and inactive forms of enzyme, E* represents a metastable

form of enzyme that is in equilibrium with Eact, and k represents the rate constant for the

conversion from E* to Ein. E* was produced as a result of ice formation, therefore

conversion from E* Ein _{involved only a few breakages of chemical interactions. For}

these reasons, the maximal inactivation was observed at the midpoint temperature, i. e. -20 oC, due to high concentration of E* and the rate of inactivation was still significant. When the enzyme was stored at -2 oC, no significant inactivation was observed because there was not enough E*. At -60 oC, most of the enzyme was in the E* form and k had become so low that the inactivation was not detectable anymore.

The freezing-inactivation study by Curti et al. was done early before the pH shift in storage buffers was investigated extensively. However, the authors also tested the effect of different buffers on the inactivation of LAAO during freezing. Lower inactivation rates were observed when sodium or potassium phosphate was used as buffers instead of Tris. The authors proposed that phosphates protected the enzyme from freezing inactivation.