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Using JPF1 Format LOW TEMPERATURE PHYSICS VOLUME 25, NUMBER 10 OCTOBER 1999 Low-temperature experimental studies in molecular biophysics: a review Yu. P. Blagoi, G. G. Sheina, A. Yu. Ivanov, E. D. Radchenko, M. V. Kosevich, V. S. Shelkovsky, O. A. Boryak, and Yu. V. Rubin B. Verkin Institute for Low Temperature Physics and Engineering, National Academy of Sciences of the Ukraine, 310164 Kharkov, Ukraine*͒ ͑Submitted April 7, 1999͒ Fiz. Nizk. Temp. 25, 1003–1020 ͑October 1999͒ The enormous contribution of Academician Boris I. Verkin in laying the foundation of the biophysics research school in Kharkov are recalled in the Jubilee year commemorating his 80th birthday. This review describes the development and realization of his ideas during the last two decades at Molecular Biophysics Department of the Institute for Low Temperature Physics and Engineering ͑ILTPE͒ in Kharkov. Main results of the studies of physical and chemical properties of biopolymer fragments and biologically active compounds using methods of low- temperature electron–vibrational spectroscopy, low-temperature secondary-emission mass spectrometry, and low-temperature luminescence spectroscopy are presented. © 1999 American Institute of Physics. ͓S1063-777X͑99͒00110-3͔ 1. INTRODUCTION important economic programs on the application of cryo- genic technologies in agriculture, food industry, medicine, The multi-faceted talent of Academician Boris and fabrication of medical and cryosurgical instruments.5,6 Ieremievich Verkin was manifested, among other things, in At the behest and under the guidance of B. I. Verkin, his interest in the application of physical ideas, techniques new experimental and theoretical methods developed earlier and methods for studying the physics of biological mol- in the physics of condensed state and low temperatures at ecules. His untiring organizational activity over the entire ILTPE were applied for studying the physical properties of span of his creative lifetime culminated in the foundation of biomacromolecules and their components. Beginning from the biophysical research school in Kharkov, which won 1969, various problems in biophysics were studied in several widespread recognition in the erstwhile USSR as well as departments of ILTPE under the supervision of leading spe- abroad. Several biophysics research departments were cialists in various branches of physics, e.g., physics of the opened at the Institute for Low Temperature Physics and liquid state ͑head of department Yu. P. Blagoi͒, physics of Engineering ͑ILTPE͒ at the end of 1960’s. The chair of mo- biological molecular crystals ͑head of department B. Ya. lecular and applied biophysics at the Radiophysics Faculty of Sukharevskii͒, tunnel spectroscopy ͑head of department I. K. the Kharkov State University was instituted in 1978, and Yanson͒, molecular interaction of biomolecules ͑head of de- Boris Ieremievich was its head for several years. In 1972, the partment L. F. Sukhodub͒, theory of the properties of bio- Institute of Cryobiological and Cryomedical Problems was logical macromolecules ͑head of department A. M. opened in Kharkov by the Ukrainian Academy of Sciences Kosevich͒, and theory of superconductivity ͑head of depart- with the support of B. I. Verkin. Kharkov became the center ment I. O. Kulik͒. These investigations are being carried out and meeting point of scientific fraternity comprising special- even today in the department of molecular biophysics headed ists in the field of biophysics. The Departments of Biophys- by Yu. P. Blagoi.4 New unorthodox and unique techniques in ics at the Institute of Radiophysics and Electronics and IL- tunnel spectroscopy, temperature-dependent field mass- TPE of the Ukrainian Academy of Sciences organized seven spectroscopy, low-temperature quartz resonator, vibrational All-Union conferences on the ‘‘Spectroscopy of Biopoly- spectroscopy of molecules isolated in the host matrix of so- mers,’’ five seminar-cum-schools on the biophysics of lidified inert gases, and luminescence spectroscopy were de- nucleic acids, and two schools on the application of mass veloped and successfully used for solving a number of prob- spectrometry in biology and medicine. The Institute of Cryo- lems. Many new results were obtained from the molecular biological and Cryomedical Problems organized several In- biophysics research with active participation, persistent inter- ternational Conferences on ‘‘Achievements and Prospects of est and support of Boris Ieremievich. Growth in Cryobiology and Cryomedicine Research.’’ Aca- Conformational and phase transitions in polymer DNA’s demician B. I. Verkin was a member of the Organizing Com- were studied experimentally and theoretically. The interac- mittees of these scientific forums. tion of native DNA with synthetic oligonucleotides with Under the guidance of B. I. Verkin, scientists at ILTPE heavy metal ions was investigated. The heat capacity of completed several academic projects on fundamental re- DNA and proteins was studied for the first time in the tem- search in the field of biopolymer physics. The results of these perature interval 4.2–400 K, and the dynamics of DNA con- investigations were published in several hundred scientific sidered in the glass-type model. A series of pioneering works papers,1 generalized in monographs,2–4 and used in many was aimed at studying the energetics of molecular interaction 1063-777X/99/25(10)/13/$15.00 747 © 1999 American Institute of Physics 748 Low Temp. Phys. 25 (10), October 1999 Blagoi et al. of nitrogenous bases simulating intramolecular interaction in DNA and RNA, hydration of bases, evaluation of the contri- bution from various types of interactions to the stabilization of the DNA molecular structure. The inelastic tunnel spec- troscopy method was used to study the energy spectra of nitrous bases. The interaction of DNA and its components with pharmacological, chemical and therapeutical prepara- tions and dyes was studied and the molecular mechanism of action of many anticancerous preparations was determined. Many inventions associated with the fabrication of cryo- surgical instrument and application of cryogrinding and sub- limative drying in food industry were patented.1 Over the years, the research activity started by B. I. Verkin has continued to develop. Special attention has been paid to the use of experimental low-temperature technology. For example, the method of matrix isolation was consider- ably modified for investigating thermally unstable molecules, and a new method of low-temperature mass spectroscopy FIG. 1. IR Fourier spectra of uracil in different states of aggregation: ϭ 14 ͑ ͒ ϭ involving bombardment of fast atoms was developed for 1–gaseous phase, T 570 K curve 1 , amorphous uracil film T 12 K ͑curve 2͒, 3—Ar matrix for molar ratio Mϭ1:1000, Tϭ12 K, D is the studying frozen aqueous solutions of biologically active optical density and ␯ the wave number ͑curve 3͒. compounds. The present review is aimed at describing the main results obtained with the help of the above-mentioned methods of biophysical research. spectroscopy of matrix isolation in comparison with the gas- 2. LOW-TEMPERATURE HIGH-RESOLUTION OPTICAL eous phase spectroscopy is its high sensitivity and lower SPECTROSCOPY OF ISOMERIC AND CONFORMATIONAL temperatures of evaporation of the thermally unstable bio- TRANSITIONS IN NUCLEOTIDE BASES AND AMINO organic materials under investigation. The required amount ACIDS of the substance is accumulated over a long time in the ma- The application of modern methods of low-temperature trix. At low temperatures, the matrix acts as a trap for vari- experiment, which are not conventional for molecular biol- ous isomers of the evaporating substance. The efficiency of ogy studies, has shed new light on the properties of biologi- this trap depends on the height of the barriers between en- cal objects. Many important questions concerning the storage ergy states of isomers and temperature of the matrix. Hence and reproduction of genetic information are connected with the temperature must be below 10 K for the investigated the isomeric nature of the simplest fragments of biopolymers low-barrier conformational isomers. in various molecular surroundings. Spectroscopic methods In order to realize the potentialities of the modern tech- are the most effective among the wide range of physical nique, two universal setups were developed. One of them is methods used for studying molecular isomerism. In many based on the modernized IR spectrophotometer Specord-IR cases, the low-temperature matrix isolation spectroscopy of- 75 and the UV spectrophotometer Hitachi-M65. The other is fers the only possibility of recording isomers of isolated mol- based on the evacuated Fourier spectrometer FS-01. Unlike ecules. This technique was employed for the first time in the the widely used closed-cycle microrefrigerators which also biophysics department of ILTPE in 19817–10 and was devel- lower the temperature to 10 K, we cooled multiple position oped side by side with other European and American re- holders of optical substrates by using helium cryostats de- search centers,11–13 thus laying the foundations of a new signed at the experimental construction bureau of ILTPE, trend in the matrix isolation spectroscopy, viz., the low- which were capable of cooling the substrate to 3 K and pro- temperature spectroscopy of isolated bio-organic molecules. viding several matrix samples. Under conditions of matrix isolation, the method of low- In addition to the traditional Ar, Kr, and Xe matrices, we temperature
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