Effect of Low Temperatures on Protein Systems


  • Oleg A. Nardid Department of Cryobiophysics, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov




low temperatures, biomacromolecules, dehydration, structural and conformational changes, cryoprotection


The review concerns the main issues of preserving the structure and function of proteins either isolated or as a part of biological tissues during hypothermic and cryogenic storage. The role of biomacromolecules’ dehydration, as a consequence of water freeze-out in proteins cryodamage during freeze-thawing is discussed, as well as peculiarities of protein low-temperature denaturation, associated with entropic nature of hydrophobic interactions. Low temperature injuries in biomacromolecules, their
reversibility with considering the role of water in structure instability of cooled or frozen biopolymers are described. Potential ways to protect biomacromolecules during freezing are considered.

Probl Cryobiol Cryomed 2014; 24(2):83-101.


Aksyonov S.I. Water as a regulator in biological systems. Studia Biophys 1981; 258(1): 37–38.

Askochenskaya N.A., Aksyonov S.I. Water structure and its role in biological systems. Uspekhi Sovr Biologii 1972; 73(2): 288–306.

Arаki N. Freezing injury in mitohondrial membranes. 1. Susceptible components in the oxidation systems of frozen and thawed rabbit liver mitochondria. Cryobiology 1977; 14(2): 144–150.

Araki N. Inactivation of mitohondrial-oxyglutarate dehydrogenase cоmplex as a result of phospholipid dehydratation induced by freeze-thawing. Cryobiology 1977; 14(2): 151–159. CrossRef

Beinert H., Hansen R.E., Hartrell C.H. Kinetic studies on cytochrome–C–oxidase by combined EPR and reflectance spectroscopy after rapid freezing. Biochim Biophys Acta 1976; 423(2): 339–356. CrossRef

Belous, A.M., Bondarenko, V.A. Structural changes in biological membranes during cooling. Kiev: Naukova Dumka; 1982.

Belous A. M., Grischenko V.I. Cryobiology. Kiev: Naukova Dumka; 1994.

Berliner L., editor. Spin labeling. Theory and applications. New York: Academic Press; 1976.

Brandts J.F. Protein conformational changes in water and mixed solutes. In: Timasheff S.N., Fasman G., editors. Structure and Stability of Biological Macromolecules. New York: Marcel Dekker Inc.; 1969, p. 213.

Burstein, E. A. The study of rapid dynamics of protein structures using the intrinsic fluorescence method. In: Burstein E.A., editor. Equilibrium dynamics of native protein structure. Puschino: Center of Biological Research, RAS; 1977, p. 60–83.

Burstein, E. A. The intrinsic luminescence of protein as the method for studies of rapid structural dynamics. Molecular Biologiya 1983; 17(3): 455–467.

Cao E., Chen Y., Cui Z., Forster P.R. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioenerg 2003; 82(6): 684–690. CrossRef PubMed

Сhilson O.F., Costello L.A., Kaplan N.O. Effect of freezing on enzymes. Fed Proc 1965; 24(2): 55–65.

Chirgadze, Yu. N., Ovsepyan A. M. Role of water in peptide structures mobility. Investigation of conformational changes during hydration. Biofizika 1972; 17(4): 569–574.

Chollot R., Anderson L.L. Conformational changes associated with the reversible cold inactivation of riboso-1,5-dyphosphate-carboxylase-oxygenase. Biophys Biochem Acta 1977; 482(1): 228–240.

Cleland K.A.P. Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric b-galactosidase. Arch Biochem Biophys 2000; 384(2): 398–406. CrossRef

Darbyshire B. The influence of dehydration on catalase stability: A comparison with freezing effects. Cryobiology 1974; 11(2): 148–151. CrossRef

Darbyshire B. The results of freezing and dehydration of horseradish peroxidase. Cryobiology 1975; 12(3): 276–281. CrossRef

Dias C.L., Ala-Nissila T., Wong-Ekkabut J. et al. The hydrophobic effect and its role in cold denaturation. Cryobiology 2010; 60(1): 91–99. CrossRef PubMed

Duzu P. Cryobiochemistry. Moscow: Mir; 1980.

Eckhardt B.M., Oeswein J.Q., Bewley T.A. Effect of freezing on aggregation of human growth hormone. Pharm Res 1991; 8(11): 1360–1364. CrossRef PubMed

Echole E. H., Anderegg J. W. An X-ray scattering investigation of the urea denaturation of BSA. J Am Chem Soc 1960; 82(10): 5085–5092.

Eytan G.D., Carroll R.C., Schats G. et al. Arrangement of the subunits in solubi1ised and membrane–bound cytochrome oxidase from bovine heart. J Biol Chem 1975; 260(22): 8589–8603.

Falko O.V, Prokopyk O.S., Volina V.V. et al. Application of placental blood serum to correct disordered morphofunctional state of adrenal glands at experimental atherosclerosis. Problems of Cryobiology and Cryomedicine 2013; 23(1): 75–83.

Falko O.V., Zemlianskykh N.G., Lipina O.V. et al. Modification of placental blood serum proteins induced by low temperatures. Biochemistry. Supplement Series Biomedical Chemistry 2012; 6(2): 194–202.

Franks F. Water and aqueous solutions at subzero temperatures. NY: Plenum Press; 1982: 484.

Frolova S.A. Low temperature influence on plant protease inhibitor system [dissertation]. Petrozavodsk, 2008.

Fedorov B.A. On the determination of globular protein volume in solution by small-single X-ray scattering. Biopolymers 1981; 20(3): 621–624. CrossRef

Fink A.L. Cryoenzymology: The use of sub-zero temperatures and fluid solutions in study of enzyme mechanisme. J Theor Biol 1976; 61(3): 419–445.

Fink A.L., Cartwright S.L. Cryoenzymology. CRC Gut Rev in Biochem 1981; 11(2): 145–207.

Fishbein W.N., Griffin J.L. External-internal ice and functional-structural damage in mouse liver mitochondria as a function of cooling rate. Cryobiology 1975; 12(6): 369–673.

Fishbein W.N., Griffin J.L. Parameters of freezing damage to mouse liver. Cryobiology 1976; 13(5): Р. 542–556.

Fishbein W.N., Stouell R.E. Studies of the mechanism of freezing damage to mouse liver using a mitochondria enzyme assay. II. Comparison of slow and rapid cooling rates. Cryobiology 1969; 6(3): 227–234.

Fitzgerald S.P., Campbell J., Lamont J.V. et al. Stabilization of enzymes during freezing. US Patent 6,579,707. 2003.

Fung B.M., Wei Sh.C. The effect of alkali and alkaline each salt on the structure and hydrated collagen fibers as studies by elaterium NMR. Biopolymers 1973; 1(5): 1053–1065.

Fung B.M., Witschel J., McAmis L.L. The state of water on hydrated collagen as studies by pulsed NMR. Biopolymers 1974; 13(9): 1767–1776. CrossRef PubMed

Gabuda S.P., Gaidash A.A., Drebushak V.A. et al Refinement of NMR data of the structure of combined water in collagen with use of scanning calorymetry. J Struct Chem 2005; 46(6): 1174–1176. CrossRef

Gislefoss R.E., Grimsrud T.K., Morkrid L. Long-term stability of serum components in the Janus Serum Bank. Scand J Clin Lab Invest 2008; 68(5): 402–409. CrossRef PubMed

Gomes G., Pikal M.J., Rodriges-Hornedo N. Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solution. J Pharm Sci 2001; 18(1): 90–97.

Gordienko E.A., Pushkar N.S. Physical grounds of low temperature preservation for cell suspensions. Kiev: Naukova dumka; 1994.

Gorobchenko O.Ð., Gerodes A.G., Nardid O.A. et al. Dielectric properties of human ovary follicular fluid at 9.2 GHz. Bioelectrochemistry 2010; 79(2): 193–197.

Grasch L., Noak F. NMR relaxation investigation of water mobility in aqueous bovine serum albumin solution. Biophys Biochem Act 1976; 453(1): 218–232.

Grischenko V.I. Achievements in cryobiology and cryomedicine for national health. Problems of Cryobiology 2008; 18(3): 269–274.

Grischenko V.I., Goltsev A.N. Transplantation of the products of embryofetoplacental complex. From understanding of me-chanism of the effect to increasing the efficiency of application. Problems of Cryobiology 2002; (1): 54–84.

Grischenko V.I., Gerodes A.G., Petrushko M.P. et al. Application of human follicular fluid at the culturing stage of gametes and embryo in IFV program. Russian Journal of Human Reproduction 1999; (6): 43–46.

Gulevsky A.K., Relina L.I. Molecular and genetic aspects of protein cold denaturation. CryoLetters 2013; 34(1): 62–82.

Hey M.J., Cdongh J.M. Ion effects on macromolecules in aqueous solution. Nature 1976; 262(5571): 807–809. CrossRef

Hostmark A.T., Glattre E., Jellum E. Effect of long-term storage on the concentration of albumin and free fatty acids in human sera. Scand J Clin Lab Invest 2001; 61(6): 443–447. CrossRef PubMed

Ilder D.R., Freemen H.C. Stability to freezing of steroidbinding proteins in fish plasma. Comp Biochem and Physiol Biochem 1973; 44(1): 179–183. CrossRef

Ishivata Sh. Freezing of action: Reversible oxidation of a sulfhydryl group and structure change. J Biochem 1976; 80(3): 595–609.

Janin J. Surface and inside volumes in globular proteins. Nature 1979; 277(5696): 491–492. CrossRef PubMed

Juul P. Stability of plasma enzymes during storage. Clin Chem 1982; 257(10): 57–51.

Kahn P.C., Schanvede J.N., Ippalito A.M. et al. Volume changes of globular protein association. Biophys J 1980; 32(1): 86–87. CrossRef

Kauzmann W., Moore K., Schulz D. Protein densities from X-ray crystallographic coordinates. Nature 1974; 248(5447): 447–449. CrossRef PubMed

Kawato S., Jochida S., Orii Y. Nanosecond time-resolved fluo-rescence investigations of temperature-induced confor-mational change in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems. Biochem Biophys Acta 1981; 634(1): 85–92.

Keshavarz E., Nakai S. The relationship between hydrophobicity and interfacial tension of proteins. Biochem Biophys Acta 1979; 576(2): 269–279.

Khenokh Ðœ.Ð., Persina V.P. Formation of hybrid enzymes under the action of profound cooling. Kriobiologia i kriomeditsina 1980; (7): 98–101.

Khenokh Ðœ.Ð., Persina V.P., Latinskaya Ye.Ðœ. Influence of deep freezing on protein solutions. Tsitologiya 1966; 8(6): 769–772.

Khurgin Iu.I., Sherman F.B., Tusupkaliev U. Isotherms of globular protein hydration under dynamic conditions. Biokhimiia 1977; 42(3): 490–498.

Klotz I.M. Comparison of molecular structure of proteins: Helix content, distribution of apolar residues. Arch Biochim Biophys 1970; 138(4): 704–706.

Lumry R., Biltonen R. Thermodynamic and kinetic aspects of protein conformations in relation to physiological function. In: Timasheff S., Fasman G., editors. Structure and stability of biological macromolecules. Moscow; 1973.

Leonov B.N., Nardid О.Ð., Moiseyev V.Ð. The influence of solvent composition on the serum albumin structure in solution. Proceedings of the 5th All-USSR Conference on Spectroscopy of Biopolymers; Oct 2–4; Kharkov, 1984. p. 142–143.

Lichtenstein G. I. The method of spin labels in molecular biology. Moscow: Nauka; 1974.

Lugovoy V.I. Primary mechanisms of enzyme cryodamage. In: Actual problems in cryobiology. Кyev; 1981. p. 15–41.

Lugovoy V.I. Primary mechanisms of enzyme cryodamage. Proceeding of the 2nd All-Union Conference on Theoretical and Applied Problems of Cryobiology; 1984 Oct 9–11; Kharkiv; 1984. p. 32.

Lugovoy V.I., Zolochevskaia L.I., Dziuba A.N. Activity of certain cytoplasmic enzymes in the exocellular medium of the bone marrow preserved at low temperature. Problemy Gematologii i Perelivaniia Krovi 1975; 21(9): 10–12.

Lugovoy V.I., Moiseyev V.A. Low temperature influence on soluble enzymes. Itogi Nauki i Tekhn VINITI ÐS SU. Ser Biofizika 1979; 9: 53–79.

Market C.L. Lactate degidrogenaze izozymes: Dissociation and recombination of subunits. Science 1963; 140(3573): 341–350.

Markosyan K.A., Pogosyan G.G., Paityan N.A. et al. Interaction of inorganic anions with copper atoms of cytochrome oxidase. Biokhimiya 1979; 44(5): 844–848.

Milson T.J., Keller R.H. The variable effects of cryopreservation on peripheral blood mononuclear populations. J Clin Labor Immunology 1982; 7(1): 205–213.

Moiseev V.A., Mank V.V., Zinchenko V.D. Hydration and dehydration properties of hemoglobin. In: Mechanisms of Crydamage and Cryoprotection of Biological Structures. Кiev: 1977. p. 44–47.

Moiseev V.A., Morozova T.F., Mikulinsky Yu.E. Influence of salt hyper concentration on horse immunoglobulin conformational state. Kriobiologia i Kriomeditsina 1980; (7): 51–52.

Morozova T.F., Grek A.M., Koptelov V.A. et al. Changes in spectral properties of bovine serum albumin in concentrated solutions of sodium chloride. Kriobiologia i Kriomeditsina 1982; (10): 22–27.

Morozova T.F., Moiseyev V.A. Salt influence on horse immunoglobulin stability. Kriobiologia i Kriomeditsina 1982; (10): 27–29.

Mosolov V.V., Sokolova E.V. Interaction of glycols and glycerin with the active center of trypsin and chymotrypsin. Doklady Academy of Sciences of USSR 1972; 207(1): 91–93.

Moshko Yu.A. Cryopreservation of cord blood serum, evaluation of its biological activity and clinical efficiency in therapy of chronic salpingoophorites [dissertation]. Kharkov; 2003.

Nardid O.A., Dyubko T.S., Repina S.V. A comparative study of the effect of freeze-thawing on peripheral and integral membrane proteins. Cryobiology 1997; 34: 107–113. CrossRef PubMed

Nardid O.A., Pogozhykh D.N., Repina S.V. et al. Influence of low temperature storage on the properties of human placenta. J Exp Integr Med 2012; 2(3): 213–217. CrossRef

Nardid E.O. Biological activity of cord blood serum in experimental model of posthysteroctomic syndrome [dissertation]. Kharkov; 2010.

Nardid E.O., Naumenko E.I., Rozanova E.D. et al. Effect of freezing regimens on protein aggregation of human cord blood serum. Problems of Cryobiology 2006; 16(3): 311–315.

Nardid E.O., Rozanova E.D., Tsymbal L.V. et al. Effect of freezing on cord blood serum proteins. Biophysics 2009; 54(5): 881–886. CrossRef

Nardid E.O., Tsymbal L.V., Nardid O.A. Influence of cryopreservation regimens on dynamics of water-protein system of human cord blood serum. Problems of Cryobiology 2005; 15(3): 544–545.

Nardid O.A. Intra– and intermolecular forces and their role in cryodamage and cryoprotection of biological structures. [dissertation]. Kharkov; 2012.

Nardid O.A., Gorobchenko O.A, Nikolov O.T. et al. Examination of low temperature influence on cord blood serum by microwave dielectric method. Fiziologichnyi Zhurnal 2005; 51(5): 56–60.

Nasonov E.L., Vinogradov V.A. Opioid peptides as regulators of the immune system. Biull Vsesoiuznogo Kardiol Nauchn Tsentra AMN SSSR 1987; 10(1): 3–10.

Naumenko E.I., Rozanova E.D. Studying of isolated cytochrome oxidase cryodamage mechanisms. Second All-USSR conference on theoretical and applied problems of cryobiology; 1984 Oct. 9–11; Kharkiv, Ukraine.

Nedev K.N., Khurgin Yu. I. A study of the surface layer of a protein globule. Hydration of a-chymotrypsin molecule. Mol. Biol. 1975; 9: 761–767.

Nikolchenko A.Ju. Investigation of the influence of low temperature on the a2-globulin group proteins and transaminases of the human donor blood [dissertation]. Kharkov; 1999.

Oboznaya E. I., Markova O.P. Investigation of terminal link of cytochrome oxidase chaine of cytochrom oxidase in bone marrow cells after deep cooling. Kriobiologia i Kriomeditsina 1975; (1): 60–62.

Pasynskiy Ð.G., Elpiner I.E. About dependence of protein hydration on pH and environmental temperature. Doklady Academy of Sciences of USSR 1955; 105(6): 1296–1299.

Pawelek P.D., Cheahl J., Coulombel R. et al. The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiometrically conserved active site. EMBO J 2000; 19: 4204–4215. CrossRef PubMed

Pogozhikh D.N., Rozanova E.D., Nardid O.A. Change of properties of human placenta aqueous-saline extracts during low temperature storage. Problems of Cryobiology 2008; 18(1): 22–26.

Polyak R. Study of immunoglobulin spatial structure. In: Immunoglobulins: Мoskow; 1981. p. 9–15.

Ponnuswamy P.K., Prabhakaran M., Manavalan P. Hydrophobic packing and special arrangement of amino acid residues in glo-bular proteins. Biochim Biophys Acta 1980; 623(2): 301–306. CrossRef

Permakov E.A., Bursten E.A. Relaxation processes in frozen aqueous solution of proteins, temperature dependence of fluorescence parameters. Studia Biophis 1975; 51(2): 91–103.

Pfeil W.P., Privalov, P.L. Conformational changes in proteins. In: Biochemical Thermodynamics: Moscow; 1982; p. 5–139.

Rozanova, E.D., Moiseyev, V.A., Naumenko, E.I. The effect of freeze-thawing on the structure and function of cytochrome oxidase. Ukr Biokhim Zhurn 1979; 57(1): 61–64.

Ruegg M., Moor V., Blance B. Hydration and thermal denaturation of g-actoglobulin: a calorimetric study. Biochem Biophys Acta 1975; 400(2): 334–342.

Sarciaux J.M., Mansour S., Hageman M.J. et al. Effect of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying. J Pharm Sci 1999; 88(12): 1354–1361. CrossRef PubMed

Scrutton M.C., Utter M.P. Pyruvate carboxylase. J Biol Chem 1965; 240(1): 1–9.

Sharon A., Levitt J. The effect of cryoprotective agents on intermolecular S–S formation during freezing on thiogel. Cryobiology 1967; 4(2): 85–89. CrossRef

Shayo B. ESR assay of changes in catalase arising during freezing and sublimation. Kholodovaya Tekhnika 1976; (1): 44–45.

Sherman J.K. Correlation in ultrastructural cryoinjury of mito-chondria with aspects of their respiratory function. Exp Cell Res 1971; 66: 378–384. CrossRef

Smanson M., Parker L. Effect of crosslinking cytochrome-c-oxidase. Arch Biochem and Biophys 1980; 204(1): 30–40. CrossRef

Storey K. B. Biochemical adaptation for cold hardiness. Phil Trans R Soc 1990; 326(1237): 635–654. CrossRef

Sun S.P., Chang T.S., del Rosario N.O. Bovine serum albumin in lithium chloride solutions. Change in conformation. Int J Peptide Protein Res 1974; 6(1): 87–94.

Sur T.K., Biswas T.K., Ali L. et al. Anti-inflammatory and anti-platelet aggregation activity of human placental extract. Acta Pharmacol Sin 2003; 24(2): 117–125.

Tang X.C., Pikal M.J., Rodriges–Hornedo N. The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying. Pharm Res 2005; 22(7): 1167–1175. CrossRef PubMed

Tabors G. Protein alteration at low temperatures. In: Proteins at low temperatures. Washington; 1979. p. 1–27.

Troitsky G.V., Tetin S.Y., Efetov K.A. A study of IgG conformation under partial dehydration. Data of temperature-perturbation differential spectrophotometry. Dopovidi Akad Sci Ukr SSR 1984; (2): 84–95.

Tverdislov V.A., Tikhonov A.N., Yakovenko L.V. Physical Mechanisms of functioning of biological membranes. Moscow; 1987.

Vets N., Akashi H. Nuclear magnetic resonance investigation of the state of water in protein solution. J Brioche 1975; 78(1): 229–234.

Volkenstein M.V. Biophysics. Moscow: Nauka; 1981.

Whittman J.H., Rosano H.L. Effects of the freeze-thaw process on amylase. Cryobiology 1973; 10(3): 240–243. CrossRef

Wino W.W., Unicode V. Sulfhydryl groups of cytochrome oxidase. J Bioenerg 1973; 4(6): 579–590. CrossRef

Wolfe J., Bryant G., Koster K.L. What is "unfreezable water", how unfreezable is it and how much is there? CryoLetters 2002; 23(3): 157–166.

Yoog Y., Pope J., Wolfe J. Freezing stresses and hydration of isolated cell waters. Cryobiology 2003; 46(3): 271–276. CrossRef

Yu N.T., Jo B.H. Comparison of protein structure in crystals and in solution by laser Raman scattering. II. Ribonuclease and caboxypeptidase. J Am Chem Soc 1973; 95(15): 5033–5037.

Yu Z., Gacia A.S., Jonston K.P. et al. Spray freezing into liquid nitrogen for highly stable protein nanostructured micro-particles. Eur J Pharm Biopharm 2004; 58(3): 522–537.

Zhmakin A.I. Physical aspects of cryobiology. Uspekhi Fizicheskikh Nauk 2008; 178(3): 243–266.




How to Cite

Nardid, O. A. (2014). Effect of Low Temperatures on Protein Systems. Problems of Cryobiology and Cryomedicine, 24(2), 83–101. https://doi.org/10.15407/cryo24.02.083