Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles."

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Principles of fluorescence spectroscopy

There is a special reason for reviewing this book at this time: it is the 50th edition of a compendium that is known and used frequently in most chemical and physical laboratories in many parts of the world. Surely, a publication that has been published for 56 years, withstanding the vagaries of science in this century, must have had something to offer. There is another reason: while the book is a standard fixture in most chemical and physical laboratories, including those in medical centers, it is not as frequently seen in the laboratories of physician's offices (those either in solo or group practice). I believe that the Handbook can be useful in those laboratories. One of the reasons, among others, is that the various basic items of information it offers may be helpful in new tests, either physical or chemical, which are continuously being published. The basic information may relate

Handbook of Chemistry and Physics

The effect of iodide on the tryptophyl fluorescence of model compounds and of lysozyme was studied in order to evaluate the factors that determine the use of iodide as a selective quencher of the fluorescence of tryptophyl side chains of proteins exposed to solvent. The results with the model compounds indicate the involvement of a collisional quenching mechanism due to the agreement with the Stern-Volmer law and the proportionality of the quenching constant with To7 for indole-3-acetamide. Bimolecular rate constants, k a , calculated from measured quenching constants using available lifetime data are equal to, greater than, or less than 4-6 X lo9 M-' sec-l for uncharged, positively charged, and negaI n a preliminary study it was shown that a large fraction of the tryptophyi fluorescence of lysozyme in aqueous solution was quenched by low concentrations of iodide ion (Lehrer, lY67). It was concluded from a study of the magnitude of the quenching of fluorescence and the character of the difference fluorescence spectrum produced in the presence and absence of substrate that the fluorescence of tryptophyls exposed to solvent and located in the substrate binding site was preferentially quenched by iodide. It appeared that this technique, which can be called solute perturbation of protein fluorescence, could be used as a probe of fluorophor exposure in proteins in a manner analogous to the technique of solvent perturbation of protein absorption (Herskovits and Laskowski, 1960; Laskowski, 1966). * From the Department of Muscle Research, Boston Biomedical Research Institute, Boston, Massachusetts 021 14, and from the Department of Neurology, Harvard Medical School, Boston, Massuchusetts 02115. Receired April 22, 1971. This work was supported by grants from the National Institutes of Health (AM 11677 and HE 0581 1) and the iMass'ichuserts Heart Association (516). tively charged tryptophyl compounds, respectively. A modified version of the Stern-Volmer law was calculated for a fluorophor population with different quantum yields and quenching constants. This formulation allows the calculation of the effective quenching constant from the intercept and the slope at low iodide concentration of a F o ] M cs. l/(I-) plot. Data obtained for lysozyme indicate that for the native protein about one-half the tryptophyl fluorescence is accessible at pH 5.3 whereas all of the tryptophyl fluorescence is accessible in 6 M G d n . HCI. Information regarding the presence of charged groups near tryptophyl side chains was obtained for lysozyme by studying the dependence of the quenching on pH. More recently, studies by other workers have ~ised bromate (Winkler, 1969) and iodide (Arrio er al., 1970) to quench extrinsic fluorescence (Teale and Badley, 1970). Oxygen has also been used as a quencher of pyrenebutyric acid bound to proteins (Vaughan and Weber, 1970). Burstein (1968a) has also independently studied the quenching of tryptophyl fluorescence in model compounds by iodide. In order to learn more about the quenching mechanism and the factors which determine fluorophor exposure, various tryptophyl model compounds and a model protein, lysozyme. were used in the present study. The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity. The quenching of lysozyme fluorescence by iodide also appears to follow a similar mechanism because of the agreement obtained with a inodified version of the Stern-Volmer law which was calculated for a heterogeneous distribution of fluorophors in a protein. Effective Stern-Volmer quenching constants and values for the fractional accessible fluorescence were obtained for lyso3254 B I O C H E M I S T R Y , V O L . 1 0 , N O . 1 7 , 1 9 7 1 I O D I D E Q U E N C H I N G O F P R O T E I N F L U O R E S C E N C E zyme in 6 M Gdn.HCI, 'S M urea, and in aqueous solution at different pH's using the modified Stern--Volmer law. Values obtained are consistent with information regarding accessibility obtained by other methods. Experimental Section Muteriais. The following high-purity compounds were used as obtained from Mann Research Laboratories, New York, N. Y. : indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid, indole-3-acetamide, N-Ac-L-TrpNH?, L-TrpOEt, Gdn . HCI, and urea. L-Trp (Cyclo Chemical Corp., Los Angeles), KI , Na&03, citric acid, and NaCl (Fisher Scientific Co., Freehold, N. J.) were all of high purity and used as obtained. Indole (Fisher) and skatole (3-methylindole) (Mann) were recrystallized from methanol containing Norit A (Matheson Coleman & Bell, Rutherford, N. J.). Hepes buffer was used as obtained from Calbiochem (Los Angeles). Poly(Glug9Trp1) and poly(Lysg7Trp3) were high molecular weight random sequence copolymers kindly supplied by Dr. G. Fasman. Lysozyme from two different sources were used (twice crystallized from Worthington Biochemical Corp., Freehold, N. J., and six-times crystallized from Miles Laboratories, Elkhart, Ind.). Both preparations gave similar results. Ac3Glcn was kindly supplied by Dr. J. Rupley and glycol chitin was obtained from Miles Laboratories. Methods. Quenching measurements at constant pH were made on five solutions of a given material containing increasing amounts of K I (0-0.2 M). These were prepared by diluting stock solutions of the model compound, of KI, of NaC1, and of buffer, into volumetric flasks. NaCl was used to keep the ionic strength constant. Stock solutions of the indole compounds were used within a few days of preparation and kept in the dark at 0-5" overnight. A small amount of SO3?(ca. M) was added to the iodide solution to prevent 1 3 formation. This was necessary because Isabsorbs in the wavelength region of tryptophyl fluorescence (filter effect) and because of possible chemical reaction. The solutions were equilibrated at 25 O before the measurements. Stock solutions of lysozyme were routinely filtered through a Millipore filter (HAWP 0.45 p ) before use. pH titrations were performed in the absence and presence of iodide by adding small quantities of 0.5 M HC1 to the solution in the cuvet, which contained 2 mM Hepes and 2 mM citrate, originally pH 8, then measuring the pH and fluorescence. pH was measured with a Radiometer PHM4c meter standardized at pH 4 and 7. Fluorescence spectra and intensities were measured by exciting a t 280 nm or longer. In most cases no corrections for iodide absorption were necessary. The fluorescence of a reference (usually the 0.2 M NaC1-0.0 M K I solution) was measured just before measuring the fluorescence of each solution in order t o correct for any exciting lamp fluctuation. Fluorescence measurements were made with either an Aminco-Bowman spectrofluorometer or an instrument that employs two Jarrell-Ash 0.25-m monochromators, an EM1 9601 B photomultiplier, and either a high-pressure 200-W mercury lamp or a 150-W high-pressure xenon lamp. Low temperatures were obtained with a refrigerated water circulator attached to the sample housing. The temperature was measured by inserting a calibrated thermistor into the sample solution. Abbreviations used are: Gdn . HCI, guanidine hydrochloride; Trp, tryptophyl or tryptophan; Hepes, N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid ; Ac3GIcii. tri-N-acetyl-D-glucosamirie. The activity of lysozyme was determined by the method of Hamaguchi et a/ . (1960). The decrease in viscosity with time caused by hydrolysis of glycol chitin (2 mg/ml) by lysozyme (0.02 mg/ml) in the presence of 0.2 M NaCl or 0.2 M KI in 2 m M citrate (pH 5.5) is the basis of this method. The specific viscosity of glycol chitin solutions in Cannon viscometers at 25" was measured with time after a small volume of lysozyme was added. The slope of the approximately linear viscosity decrease between 1 and 10 min was used as a measure of activity. The optical rotatory dispersion and circular dichroism spectra of lysozyme (0.95 mg/ml) in 0.2 M NaCl or in 0.2 M KI , 2 mM citrate (pH 5.2) were measured in a 1-cm cell with a Jasco spectropolarimeter. The absorbance of Iprevented measurements below 265 nm. Difference spectra were either measured with a Cary 15 or a Beckman DK spectrophotometer using mixing cells (Pyrocell, Inc., N. Y . ) . The total absorption over the wavelengths scanned was always below 2.2. The low-temperature studies were performed with a Beckman D K using a refrigerated sample holder.

Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.

(2003). Hepatotoxicity and Mechanism of Action of Haloalkanes: Carbon Tetrachloride as a Toxicological Model. Critical Reviews in Toxicology: Vol. 33, No. 2, pp. 105-136.

Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model.

International programme on chemical safety

A series of 15 halogenated hydrocarbons of industrial and environmental importance were tested for carcinogenicity by chronic administration by one or more routes in Ha:ICR Swiss mice. Not all compounds were tested by the four routes of administration used. Allyl chloride, 1,2-dibromo-3-chloropropane, and vinylidene chloride were active as skin tumor initiators in the two-stage carcinogenesis assays; phorbol myristate acetate was used as a promoter. 1,2-Dibromoethane was the only compound that induced a significant incidence (p less than 0.05) of skin papillomas, skin carcinomas, and lung tumors by repeated skin application. 1,2-Dichloroethane, 1,1,2,2-tetrabromoethane, and 1,2-dibromo-3-chloropropane induced lung and/or stomach tumors by repeated skin application. Two compounds showed sarcomagenic activity by sc injection; they were cis-1,3-dichloropropene and 2-chloropropanal. By intragastric intubation, 1-chloropropene and 2-chloropropanal induced significant numbers of stomach tumors. Vehicle, no-treatment, and positive control groups were included in these tests. The following compounds were also tested by one or more of the four routes but were inactive by the criteria used; i.e., they showed P = 0.05 or greater than 0.05: trichloroethylene, tetrachloroethylene, hexachlorobutadiene, chloroacetaldehyde, 1-chloropropene oxide (cis and trans), and trichloroethylene oxide.

Carcinogenicity of Halogenated Olefinic and Aliphatic Hydrocarbons in Mice

Solvent Effects in the Fluorescence of Indole and Substituted Indoles1

Structure and tumor-promoting activity of anthralin (1,8-dihydroxy-9-anthrone) and related compounds.

A biologically potent, tumor-promoting phorbol ester from Croton tiglium L. induces the appearance of transformed clones in a population of contact-inhibited fibroblasts of the mouse cell line 3T3. Mixed populations of cells with 10,000 3T3 cells and 100 virus-transformed cells were exposed to the phorbol ester and, in comparison with untreated cells, showed a marked increase in the numbers of transformed clones that grew. Exposure of 3T3 cultures to the carcinogen 7,12-dimethylbenz(a)anthracene alone or prior to exposure to the phorbol ester did not cause any increase in the number of transformed clones.

https://science.sciencemag.org/content/sci/157/3795/1443.full.pdf

Phenotypic Expression of Transformation: Induction in Cell Culture by a Phorbol Ester

Studies on the mechanism of the carcinogenic action of vinyl chloride involve the consideration of whether the chloroolefin is the active carcinogenic agent or whether activated carcinogenic metabolites are involved. Therefore, it is instructive to consider the same question in earlier studies with a wide variety of chemical carcinogens. No attempt is made here to give a review of the literature on this subject, but only an indication of what appears to us to -be the current thinking in this area. There is a considerable body of evidence which supports the view that many well-known carcinogenic agents such as nitrosamines, aromatic amines, azo dyes and aromatic hydrocarbons have to be metabolized in situ to their activated carcinogenic intermediates.', * Although not firmly established for all of these carcinogens, much of the evidence suggests that the intermediate agents are alkylating in nature, i.e., electrophilic agents. Such electrophiles undergo a variety of reactions with nucleophiles in vivo and it is reasonable to assume that one or more of these nucleophilic reactions is or are responsible for the carcinogenicity of the parent compounds. These carcinogens can, therefore, be referred to as indirect-acting alkylating carcinogens. Many of these compounds are also capable of physical binding with tissue constituents, This noncovalent binding of aromatic hydrocarbons to nucleic acids, for example, has been extensively studied in many laboratories.' Most researchers in chemical carcinogenesis now believe that such physical binding is not related to the critical interactions involved in carcinogenesis by aromatic carcinogens. However, these studies and conclusions derived from them do not exclude the possibility that physical binding with tissue constituents plays an important role for some chemical carcinogens. The direct-acting alkylating carcinogens include such classes as the epoxides, p-lactones, and halo ethers. These agents do not have to be metabolized to exert their carcinogenic effect and for this reason they are usually carcinogenic only at the site of administration in laboratory experiments. There are some exceptions, e.g., propane sultone, which is chemically less reactive than other direct-

Benjamin L. Van Duuren

Papers

Carcinogenicity of Halogenated Olefinic and Aliphatic Hydrocarbons in Mice

Solvent Effects in the Fluorescence of Indole and Substituted Indoles1

Structure and tumor-promoting activity of anthralin (1,8-dihydroxy-9-anthrone) and related compounds.

Phenotypic Expression of Transformation: Induction in Cell Culture by a Phorbol Ester

On the possible mechanism of carcinogenic action of vinyl chloride