The term "biotin" should now replace several old names, to wit: coenzyme R, protective factor X, the protective factor against egg white injury, the vitamin H and a part of the Bios II b or the adsorbable factor of the yeast growth promoting substance. It was isolated and from the Bios II b factor of yeast as a methyl ester of the empirical composition C 11 H 18 O 3 N 2 S and having a melting point of 166°-167° when highly purified. Saponification with cold alkali gives free biotin of the empirical composition C 10 H 16 O 3 N 2 S and melting at 230°-232°. Biotin is a simple monocarboxylic acid, a derivative of valeric acid. The nitrogen atoms form a urea structure which can be opened up with a loss of one carbon to form a diaminocarboxylic acid, from which in turn one can reform biotin by reaction with phosgene. The sulfur is functioning in a thioether structure, since a sulfone can be formed by oxidation. The most probable structure is that of 2′-keto-3,4-imidazolido-2-tetrahydrothiophene-n-valeric acid.
Looked upon as an active principle of yeast growth biotin is active even at a concentration of one part in 500,000,000,000. As the active substance stimulating several species of the root-nodule bacterium Rhizobium and obtained from concentrated cultures of Azotobacter, and previously known as coenzyme R (R=respiration) it shows effects in concentration of I part in 100 billion. As Vitamin H (H for "Haut" or skin) it counteracts the effects of avidin of both raw and dried egg white to the extent of 10,000 units per milligram by a rat assay method (i.e. 0.1 gamma of biotin per rat per day for 30 days protects against egg-white injury).
The biotin may be extracted from liver, dried eggs, potato starch, fresh eggs, dried yeast, autolyzed yeast.Inactivation of biotin has been accomplished both under acid and alkaline conditions. Aeration is not effective but stronger oxidizing agents destroy activity quickly and completely. Nitrous acid inactivates without a loss of nitrogen.Biological effects of biotin other than those used in assay of the foregoing effects concern the fatty infiltration of the livers of rats on a low fat diet with high biotin. The fermentation of yeast is increased as well as its respiration and growth both aerobically and anaerobically, in the presence of plentiful nitrogen in the form of ammonia. Butter-yellow tumor formation brought on by avidin-containing diets should be counteracted by biotin.
Thursday, September 27, 2007
Biophysics scope principles methods
The domain of discussion may be indicated by defining biophysics as the application to biological research of the methods and content of the physico-mathematical sciences. "Method" is advisedly placed first. It has become clear that the experimental and theoretical procedures usually associated with physics are not the exclusive property of that science, but represent rather adequately the developed and mature form of the scientific method itself. Biophysics is the adaptation of this methodology to the problems of biology.
It appears from this that the word "biophysics" is in part a misnomer. Biophysical research is biological research with a particular emphasis--concern for the logical ordering and quantification of biological theory, for the more intimate unification of theory and experiment, for the introduction into experimental biology of techniques and measuring devices which have been developed in the so-called physical sciences, and for the reduction-when it is possible and useful, and only then-of biological problems and concepts to already existent laws and concepts of the physical sciences.
That physics--the science of matter--has in fact preoccupied itself with those objects which used to be termed "non-living" is logically accidental, not intrinsic. Taken in this sense, the "physicalizing" of biology merely means the attempt to analyze certain complex phenomenal patterns in terms of somewhat simpler patterns which have already been the object of intensive study. In this sense it is neither novelty nor heresy, but an inevitable extension of the classical biological procedure of analyzing organisms into organs, organs into tissues, tissues into cells, and cells finally into their discernible parts.
It is only fair to add that "biophysics" is usually used in a sense which contrasts it with biochemistry--that is, as a study of the physical as distinct from the chemical aspects of biological systems. This viewpoint would see viscosity, osmotic pressure, surface tension, the electrical and mechanical properties of biological systems as physical, while the composition and metabolic activities of such systems are the concern of chemistry.
This criterion has been serviceable in the past, of course; but it is so narrow as to be both cramping to research and rather difficult rigidly to maintain. Thus, the study of bioluminescence must in this view separate sharply into two parts: the analysis of the luciferinluciferase system is biochemistry, while the emission of a photon by the reduced enzyme is biophysics. It is valid to say, "For the moment, let us consider only the physical aspects of this phenomenon, and ignore the chemical and purely biological." But the moment passes quickly, and the essential inseparability of the different aspects demands the broader interpretation.
It remains to consider the best way of subdividing biophysical activity into its branches according to some specified criterion of classification. From the preceding discussion it is clear that one apparently natural division-into theoretical and experimental--is not a desirable one. The nature of biophysics, as here defined, demands the same close interweaving, mutual support, and mutual stimulus of theorizing and experimenting as is now practised so successfully in physics.
To adopt the existing fundamentum divisionis of biology is no more desirable; for the fences around bacteriology, embryology, histology, physiology, botany, and the like, in view of their haphazard historical origin, exhibit small regard either for practical utility or for logical consistency. Some of these fields are organized around a particular function, some around a particular set of structures, some around a particular class of organisms, and some are mere catch-alls for topics which do not fit handily into the remaining cells.
If we accept the cell as fundamental concept in biology, a useful though by no means perfect mode of classification suggests itself--the hierarchy: cell-parts, cells, cell-aggregates. These further subdivide in a fairly natural way, principally according to functions and activities. Thus, under Cell-parts, we think of: Protoplasmic Structure; Enzyme Systems of the Cell; Nucleus and Chromosomes; Cell Membranes and Permeability; Golgi Bodies and Mitochondria.
Under Cells would come: Growth; Form; Motion; Division; Differentiation; Metabolism; Senescence; Stimulus-Response.
Under Cell-aggregates there are similar categories: Organic Form and Differentiation; Growth; Metabolism; Senescence; StimulusResponse. These would intersect with the triple classification: Tissues; Organs; Organisms. Thus, for example, we would find Secretion under Metabolism, Organs (perhaps also under Cells, Metabolism); special senses under Stimulus-response, Organs; and the nervous and endocrine systems under Stimulus-response, Organisms. Another category under Cell-aggregates would be Populations, which would cover the material of biometrics, ecology, and much of bacteriology.
Such a topic as The Virus is not an insuperable obstacle to this arrangement; there are many good reasons for placing it under CellParts. But it is obvious from the topics named that a better method than either equal-ranking classes or a hierarchy would be a multidimensional network, in which topics may be different distances apart, but in which each topic is linked to many others directly, and to all others indirectly. Protoplasmic Structure would be close to if not precisely at the centre of such network. With the further development of biophysics, the appropriate ordering will doubtless become more evident.
Applied biophysics is exceedingly scanty. There are a few scattered medical applications, such as the gold number or its modern equivalents in the analysis of cerebrospinal fluid, the use of X-rays and radium in the treatment of cancer. But biophysics is yet too young to show applications on any large scale.
There are many fields of biology in which the rather young biophysical method has not been active, so that a sufficient amount of representative material is not available for presentation. Some topics of biophysical interest, e.g., metabolism, are still referred to biochemistry for the same reason.
It appears from this that the word "biophysics" is in part a misnomer. Biophysical research is biological research with a particular emphasis--concern for the logical ordering and quantification of biological theory, for the more intimate unification of theory and experiment, for the introduction into experimental biology of techniques and measuring devices which have been developed in the so-called physical sciences, and for the reduction-when it is possible and useful, and only then-of biological problems and concepts to already existent laws and concepts of the physical sciences.
That physics--the science of matter--has in fact preoccupied itself with those objects which used to be termed "non-living" is logically accidental, not intrinsic. Taken in this sense, the "physicalizing" of biology merely means the attempt to analyze certain complex phenomenal patterns in terms of somewhat simpler patterns which have already been the object of intensive study. In this sense it is neither novelty nor heresy, but an inevitable extension of the classical biological procedure of analyzing organisms into organs, organs into tissues, tissues into cells, and cells finally into their discernible parts.
It is only fair to add that "biophysics" is usually used in a sense which contrasts it with biochemistry--that is, as a study of the physical as distinct from the chemical aspects of biological systems. This viewpoint would see viscosity, osmotic pressure, surface tension, the electrical and mechanical properties of biological systems as physical, while the composition and metabolic activities of such systems are the concern of chemistry.
This criterion has been serviceable in the past, of course; but it is so narrow as to be both cramping to research and rather difficult rigidly to maintain. Thus, the study of bioluminescence must in this view separate sharply into two parts: the analysis of the luciferinluciferase system is biochemistry, while the emission of a photon by the reduced enzyme is biophysics. It is valid to say, "For the moment, let us consider only the physical aspects of this phenomenon, and ignore the chemical and purely biological." But the moment passes quickly, and the essential inseparability of the different aspects demands the broader interpretation.
It remains to consider the best way of subdividing biophysical activity into its branches according to some specified criterion of classification. From the preceding discussion it is clear that one apparently natural division-into theoretical and experimental--is not a desirable one. The nature of biophysics, as here defined, demands the same close interweaving, mutual support, and mutual stimulus of theorizing and experimenting as is now practised so successfully in physics.
To adopt the existing fundamentum divisionis of biology is no more desirable; for the fences around bacteriology, embryology, histology, physiology, botany, and the like, in view of their haphazard historical origin, exhibit small regard either for practical utility or for logical consistency. Some of these fields are organized around a particular function, some around a particular set of structures, some around a particular class of organisms, and some are mere catch-alls for topics which do not fit handily into the remaining cells.
If we accept the cell as fundamental concept in biology, a useful though by no means perfect mode of classification suggests itself--the hierarchy: cell-parts, cells, cell-aggregates. These further subdivide in a fairly natural way, principally according to functions and activities. Thus, under Cell-parts, we think of: Protoplasmic Structure; Enzyme Systems of the Cell; Nucleus and Chromosomes; Cell Membranes and Permeability; Golgi Bodies and Mitochondria.
Under Cells would come: Growth; Form; Motion; Division; Differentiation; Metabolism; Senescence; Stimulus-Response.
Under Cell-aggregates there are similar categories: Organic Form and Differentiation; Growth; Metabolism; Senescence; StimulusResponse. These would intersect with the triple classification: Tissues; Organs; Organisms. Thus, for example, we would find Secretion under Metabolism, Organs (perhaps also under Cells, Metabolism); special senses under Stimulus-response, Organs; and the nervous and endocrine systems under Stimulus-response, Organisms. Another category under Cell-aggregates would be Populations, which would cover the material of biometrics, ecology, and much of bacteriology.
Such a topic as The Virus is not an insuperable obstacle to this arrangement; there are many good reasons for placing it under CellParts. But it is obvious from the topics named that a better method than either equal-ranking classes or a hierarchy would be a multidimensional network, in which topics may be different distances apart, but in which each topic is linked to many others directly, and to all others indirectly. Protoplasmic Structure would be close to if not precisely at the centre of such network. With the further development of biophysics, the appropriate ordering will doubtless become more evident.
Applied biophysics is exceedingly scanty. There are a few scattered medical applications, such as the gold number or its modern equivalents in the analysis of cerebrospinal fluid, the use of X-rays and radium in the treatment of cancer. But biophysics is yet too young to show applications on any large scale.
There are many fields of biology in which the rather young biophysical method has not been active, so that a sufficient amount of representative material is not available for presentation. Some topics of biophysical interest, e.g., metabolism, are still referred to biochemistry for the same reason.
Bioluminescence
Bioluminescence is a word applied to the light emitted by various living organisms. At least 40 different orders of animals contain luminous species and two groups of plants, the bacteria, responsible for the luminescence of flesh and dead fish, and the fungi, which live in phosphorescent wood. Luminous bacteria are so small that individuals cannot be seen by their own light but colonies are visible. They are easily cultured and are nonpathogenic to man but may infect living animals, giving rise to a luminescent disease of sand-fleas, shrimps and midges, which is eventually fatal. Luminous bacteria may also live symbiotically in special organs of certain fish, notably Photoblepharon and Anomalops, of the Banda islands.
Bacteria and fungi emit light continuously day and night, while all other forms luminescence only when stimulated. The phosphorescence of the sea appears when many different kinds of small organisms are disturbed by the breaking of waves or motion of a boat. Among the groups of animals containing luminous species are flagellates, radiolaria, sponges, jelly-fish hydroids, sea pens, ctenophores, nemerteans, earthworms and many marine worms, shrimp, ostracods and copepods, myriapods, several groups of insects, monuses, squid, brittle stars, balanoglossids, tunicates and fish.
Bioluminescence is never dependent on a previous illumination of the cells or a previous radiation of any kind, nor is it connected with crystallization, friction, or rubbing, but is the result of oxidation by molecular oxygen of a definite substance produced in the luminous cell. It is a chemiluminescence. The luminous material or photogen is almost universally manufactured by living cells as granules, which may normally undergo oxidation within the cell, as in the fire-fly, (intracellular luminescence) or be extruded as a luminous slime or secretion (extracellular luminescence) as in a small ostracod crustacean, Cypridina. Most is known concerning the chemistry of extracellular luminescence, especially that of Cypridina.
In Cypridina the granules in the secretion dissolve on contact with sea water and the homogeneous luminescence is emitted by the resultant colloidal solution. Two kinds of granules are distinguishable in the cells of Cypridina, one large and yellow, the other small and colorless. In fact, in five of twenty-five different groups of luminous animals tested, it can be demonstrated that luminescence is due to two chemical substances, luciferin (yellow) and luciferase (colorless), which can be easily separated because of a difference in resistance to heating and other properties.
Crude luciferin solution is prepared by making a hot water extract of a luminous organ. Heating destroys the luciferase but does not harm the luciferin. Crude luciferase solution is prepared by making a cold water extract of a luminous organ, when both luciferin and luciferase dissolve and luminescence occurs. The extract is then allowed to stand in the air until the light disappears, evidence that the luciferin has been completely oxidized, leaving the luciferase, an enzyme, in solution. A luciferase solution, by virtue of this mode of preparation, must contain the oxidation product of luciferin as well as luciferase.
Luciferin and luciferase are quite specific. Luciferin from one animal will not luminesce if mixed with the luciferase of another luminous form unless the animals are closely related, such as two species of the same genera or two genera within the same order. Even if the color of the luminescence is different in the two different species light will appear, provided the species are closely related. In this case it is interesting to note that the color of luminescence of the resultant "cross" is determined by the animal supplying the luciferase. Luciferase must be the source of the light. It is convenient to designate the luciferins and luciferases by prefixing the name of the animal from which the substances are obtained.
Most luminous animals, if dried rapidly will again luminesce on moistening. Dried Cypridinae have been kept for 26 years without deterioration and can be used for preparing luciferin and luciferase.
Cypridina luciferin is purified by extraction of the dry Cypridinae with methyl alcohol. Ten per cent of butyl alcohol is then added and the methyl alcohol removed in vacuo. The supernatant butyl alcohol extract is chilled and benzoylated with benzoyl chloride. After fifteen minutes this solution is diluted with ten volumes of water and the new inactive benzoyl luciferin derivatives extracted with pure ether. After removing the ether in vacuo the residual liquor is hydrolyzed with hydrochloric acid in absence of oxygen. The free active luciferin is left in the acid solution and can be extracted with butyl alcohol. By repeating the benzoylation and hydrolysis, a product purified 2,000-fold, as compared with dry Cypridinae, can be obtained.
To purify luciferase it is usually sufficient to dialyze a cold, wellstirred water extract of rapidly dried, powdered Cypridinae against cold running water for twenty hours. Dialysis removes pigment, and a precipitate forms which can be filtered off. A few drops of toluene added to this solution will preserve it for months with little loss in activity if kept in a refrigerator.
Cypridina luciferin is slowly dialyzable; not destroyed by trypsin; soluble in water, absolute methyl, ethyl, and propyl alcohol but insoluble in acetone, benzene and ether; readily adsorbed on fine particles. It does not act as an antigen.
Cypridina luciferase is non-dialyzable; destroyed by trypsin; soluble in water, but insoluble in alcohols and all fat solvents; readily adsorbed on surfaces. It is capable of forming an antiluciferase when injected into the blood of a rabbit. Other luciferins and luciferases have different properties.
The oxidation of luciferin with production of light in the presence of luciferase gives an oxidation product which cannot be reduced, whereas the oxidation without luminescence by oxidants like potassium ferricyanide is reversible.
This Product is called oxidized lucifenn. The change with ferricyanide occurs in two steps, one of which is the reversible oxidation previously referred to; the second is irreversible and probably also an oxidation, although this has not been definitely demonstrated. The spontaneous oxidation of luciferin without emission of light in crude solutions (without luciferase) is probably catalyzed by traces of heavy metals in the solution and proceeds much more slowly when the luciferin has been purified. Both the non-luminescent oxidation and the luminescent oxidation undoubtedly take place simultaneously when luciferin is mixed with luciferase.
Cyanide does not affect luciferase but forms an irreversible combination with purified Cypridina luciferin. Azide combines reversibly with luciferin whereas urethane, sulfanilamide, sulphathiazol, sulphapyridine and p-aminobenzoic acid probably act reversibly to inhibit luciferase activity.
Bacteria and fungi emit light continuously day and night, while all other forms luminescence only when stimulated. The phosphorescence of the sea appears when many different kinds of small organisms are disturbed by the breaking of waves or motion of a boat. Among the groups of animals containing luminous species are flagellates, radiolaria, sponges, jelly-fish hydroids, sea pens, ctenophores, nemerteans, earthworms and many marine worms, shrimp, ostracods and copepods, myriapods, several groups of insects, monuses, squid, brittle stars, balanoglossids, tunicates and fish.
Bioluminescence is never dependent on a previous illumination of the cells or a previous radiation of any kind, nor is it connected with crystallization, friction, or rubbing, but is the result of oxidation by molecular oxygen of a definite substance produced in the luminous cell. It is a chemiluminescence. The luminous material or photogen is almost universally manufactured by living cells as granules, which may normally undergo oxidation within the cell, as in the fire-fly, (intracellular luminescence) or be extruded as a luminous slime or secretion (extracellular luminescence) as in a small ostracod crustacean, Cypridina. Most is known concerning the chemistry of extracellular luminescence, especially that of Cypridina.
In Cypridina the granules in the secretion dissolve on contact with sea water and the homogeneous luminescence is emitted by the resultant colloidal solution. Two kinds of granules are distinguishable in the cells of Cypridina, one large and yellow, the other small and colorless. In fact, in five of twenty-five different groups of luminous animals tested, it can be demonstrated that luminescence is due to two chemical substances, luciferin (yellow) and luciferase (colorless), which can be easily separated because of a difference in resistance to heating and other properties.
Crude luciferin solution is prepared by making a hot water extract of a luminous organ. Heating destroys the luciferase but does not harm the luciferin. Crude luciferase solution is prepared by making a cold water extract of a luminous organ, when both luciferin and luciferase dissolve and luminescence occurs. The extract is then allowed to stand in the air until the light disappears, evidence that the luciferin has been completely oxidized, leaving the luciferase, an enzyme, in solution. A luciferase solution, by virtue of this mode of preparation, must contain the oxidation product of luciferin as well as luciferase.
Luciferin and luciferase are quite specific. Luciferin from one animal will not luminesce if mixed with the luciferase of another luminous form unless the animals are closely related, such as two species of the same genera or two genera within the same order. Even if the color of the luminescence is different in the two different species light will appear, provided the species are closely related. In this case it is interesting to note that the color of luminescence of the resultant "cross" is determined by the animal supplying the luciferase. Luciferase must be the source of the light. It is convenient to designate the luciferins and luciferases by prefixing the name of the animal from which the substances are obtained.
Most luminous animals, if dried rapidly will again luminesce on moistening. Dried Cypridinae have been kept for 26 years without deterioration and can be used for preparing luciferin and luciferase.
Cypridina luciferin is purified by extraction of the dry Cypridinae with methyl alcohol. Ten per cent of butyl alcohol is then added and the methyl alcohol removed in vacuo. The supernatant butyl alcohol extract is chilled and benzoylated with benzoyl chloride. After fifteen minutes this solution is diluted with ten volumes of water and the new inactive benzoyl luciferin derivatives extracted with pure ether. After removing the ether in vacuo the residual liquor is hydrolyzed with hydrochloric acid in absence of oxygen. The free active luciferin is left in the acid solution and can be extracted with butyl alcohol. By repeating the benzoylation and hydrolysis, a product purified 2,000-fold, as compared with dry Cypridinae, can be obtained.
To purify luciferase it is usually sufficient to dialyze a cold, wellstirred water extract of rapidly dried, powdered Cypridinae against cold running water for twenty hours. Dialysis removes pigment, and a precipitate forms which can be filtered off. A few drops of toluene added to this solution will preserve it for months with little loss in activity if kept in a refrigerator.
Cypridina luciferin is slowly dialyzable; not destroyed by trypsin; soluble in water, absolute methyl, ethyl, and propyl alcohol but insoluble in acetone, benzene and ether; readily adsorbed on fine particles. It does not act as an antigen.
Cypridina luciferase is non-dialyzable; destroyed by trypsin; soluble in water, but insoluble in alcohols and all fat solvents; readily adsorbed on surfaces. It is capable of forming an antiluciferase when injected into the blood of a rabbit. Other luciferins and luciferases have different properties.
The oxidation of luciferin with production of light in the presence of luciferase gives an oxidation product which cannot be reduced, whereas the oxidation without luminescence by oxidants like potassium ferricyanide is reversible.
This Product is called oxidized lucifenn. The change with ferricyanide occurs in two steps, one of which is the reversible oxidation previously referred to; the second is irreversible and probably also an oxidation, although this has not been definitely demonstrated. The spontaneous oxidation of luciferin without emission of light in crude solutions (without luciferase) is probably catalyzed by traces of heavy metals in the solution and proceeds much more slowly when the luciferin has been purified. Both the non-luminescent oxidation and the luminescent oxidation undoubtedly take place simultaneously when luciferin is mixed with luciferase.
Cyanide does not affect luciferase but forms an irreversible combination with purified Cypridina luciferin. Azide combines reversibly with luciferin whereas urethane, sulfanilamide, sulphathiazol, sulphapyridine and p-aminobenzoic acid probably act reversibly to inhibit luciferase activity.
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