The analogy is not altogether apt. Catalysts are properly defined as substances that regulate the rate at which chemical reactions proceed. Some catalysts accelerate reactions that otherwise proceed slowly. Other catalysts slow reactions. Examples of both types exist in the thousands in the inorganic kingdom and within the cells and fluids of living organisms, where they are known as enzymes.

Enzymes differ from inorganic catalysts in that they are proteins and as such are large, fragile molecules built from amino acids linked end to end. Their positive identification at the turn of the present century by the German chemist Eduard Buchner opened a whole new field of experimentation that is as complex as it is fascinating. This may explain why amateurs tend to shun the enzymes. Most enzymes exist in only trace amounts and are all but impossible to isolate. But one need not isolate an enzyme in order to investigate the effects that stem from its presence. This is well illustrated by a series of experiments suggested by Henry Soloway of the department of pathology at Kings County Hospital in Brooklyn.

"To detect the presence of an enzyme," Soloway writes, "one takes advantage of the fact that an enzyme mediates a specific biochemical reaction. In other words, to demonstrate the presence of an enzyme that converts substance A into substance B, a known quantity of solution A is mixed with the solution suspected of containing the enzyme. After an appropriate interval the mixture is tested for the presence of substance B. If B is detected, the presence of the suspected enzyme is confirmed. An equal quantity of substance A, unmixed with the suspected enzyme, is kept in a separate container under the same conditions for an equal interval as a control.

"In principle the procedure is simple and fairly obvious, but putting it into practice is another matter. Enzymes are temperamental molecules. They function only in rigidly controlled environments. Maintaining such conditions in the laboratory often requires costly facilities. Temperature changes of even a few degrees exert a profound effect on them, as do slight variations in the acidity or alkalinity of the solution. Many refuse to function in the absence of trace amounts of substances called activators, and their action can be completely arrested by traces of heavy metals dissolved from laboratory glassware. Such difficulties may explain in part why the study of enzymes has failed to lure an enthusiastic following of amateurs.

"Fortunately, a few rugged enzymes have been identified that are readily available. One in particular is well suited to amateur experimentation and can be used to demonstrate most of the basic principles of enzyme reaction. It is called ptyalin and is secreted by the salivary glands. It has the function of splitting starch into sugar.

"The starch molecule is a large one, with branching chains of glucose units. Ptyalin splits off the glucose units two at a time, thereby destroying the integrity of the starch and forming numerous fragments composed of two glucose units each. To observe this action the experimenter must prepare a solution of the enzyme and one of starch, and also a system capable of detecting the amount of starch consumed or the number of twounit glucose fragments produced.

"Ptyalin is collected in saliva and cornstarch can be bought from a grocer. Iodine changes the normally milky color of starch solutions to a bluish-black, the depth of the shade increasing with the concentration of starch in solution. It therefore functions well as a detector or indicator of enzyme action.

"To collect saliva place a glass funnel lined with coarse filter paper over a glass beaker. Salivation can be encouraged by chewing a piece of paraffin of the kind used for sealing jelly. The amount of ptyalin present in saliva varies from person to person, so it is desirable to collect a pooled sample from a number of individuals. Approximately 80 milliliters should be collected and stored in a refrigerator at 40 degrees Fahrenheit. All the experiments to be described must utilize this stock solution. The concentration of ptyalin may vary substantially in different collections and the variation might introduce confusing results if tests made on one solution were grouped with those made on another.

"The starch solution is prepared by pouring one liter of boiling water over two level tablespoons of powdered cornstarch. (Distilled water should be used throughout these experiments.) After thorough mixing with a spoon or a glass stirring rod the solution is filtered through coarse filter paper (a paper napkin will do) to remove undissolved lumps of starch. The amount of starch contained in one milliliter of this stock solution is adopted as one unit of starch activity for the purposes of the experiments, and all measurements are expressed as a percentage of this unit. Since the results of the experiments are all relative, the numbers expressing Quantities are empirical; the investigator can alter them to units of any size. But by the same token measurements made with one set of solutions cannot be lumped with those of another set.

"The indicator solution is prepared by diluting 10 milliliters of 2 per cent tincture of iodine in 600 milliliters of water. The activity of the enzyme is determined by measuring the amount of light transmitted by a test tube containing the stained starch. A photometer measures the light intensity. The solution to be measured is placed between a standard source of light and a silicon solar cell. The cell converts the light to an electric current,- which actuates a milliammeter. The circuit includes a potentiometer with which to adjust the milliammeter to full scale during calibration [see illustration above]. The cell, test tube and potentiometer are assembled in a lightproof box of corrugated cardboard (or some other opaque sheet material). A lightproof partition in the box supports the test tube as shown in the accompanying drawing [below]. A slit in the two sheets of cardboard that form the partition allows light from the source to reach the solar cell. A sheet of translucent tissue paper of the type used for making tracings is placed between the test tube and the cell to diffuse the beam and so distribute the light uniformly over the active face of the cell. A 35-millimeter projector will serve as a light source, or an incandescent bulb can be built into a lightproof compartment of the box. The test tube should fit snugly against the slit facing the source so that all the light reaching the cell will pass through the test tube.

"To calibrate the photometer one prepares a series of starch standards, measuring the light transmitted by each standard and plotting the measurements. The resulting graph shows the relative variations of the meter readings with respect to the percentage of starch in solutions of unknown concentration.

"To make up the standard starch solutions for calibrating the instrument place in glass containers 20, 15, 10, five and two milliliters of the starch solution. (All glassware, including the pipettes used for transferring solutions, must be scrupulously clean.) Then add enough water to bring each of the last four containers up to 20 milliliters of starch solution. Fill a sixth container with 20 milliliters of distilled water. Mark the container with the undiluted solution 100 per cent starch and the remaining five 75, 50, 25, 1O and O per cent respectively. Next, place exactly one milliliter of each of the six standard starch solutions in each of six test tubes of equal size, add 10 milliliters of iodine indicator solution and one milliliter of water. (The milliliter of water represents the volume in which ptyalin will be contained during subsequent experiments.) Shake all test tubes gently to distribute the color evenly. Then insert the most transparent tube (the one containing no starch) in the photometer, turn on the light source and adjust the potentiometer so that the pointer of the milliammeter swings to the top of the scale. Prepare a table of two columns, one for the meter readings and the other for the percentages of starch in solution. Enter the maximum value of the meter scale in the first column and 'O' in the starch column and then, without changing the potentiometer setting or the light intensity, proceed to measure and tabulate the light transmission of the remaining five solutions. Plot the results as shown in the accompanying illustration [below]. The resulting calibration curve will be valid only for the materials in this particular experiment.

"With these preparations completed the first portion of the experiment can be undertaken. This part of the experiment will determine how the amount of starch broken down into glucose increases with time. First, clean all glassware thoroughly. Then place exactly one milliliter of standard starch solution in each of six test tubes of equal size. Using another clean pipette, transfer one milliliter of filtered saliva to each of these tubes. After 15 seconds add 10 milliliters of indicator solution to the first tube, swirl the contents to distribute the color, measure the light transmission promptly and tabulate the measurement. After 30 seconds repeat the procedure, using the second test tube of solution, and then repeat it with the remaining samples after one, two, four and seven minutes respectively. The over-all reliability of the experiment will be no greater than the accuracy with which the measurements are timed. Unless the experimenter is experienced in making measurements of this type it is advisable to run through the procedure a few times, perhaps using tubes of colored water.

"The tabulated readings are converted to percentages of starch digested by reference to the calibration graph and are then plotted against time. (Because the schedule of measurement involves constantly increasing intervals that range from seconds to minutes, the time coordinate of the graph should be divided into logarithmic intervals.) The concentration of ptyalin used in this portion of the experiment is so high that substantially all the starch is digested within approximately 30 seconds. The rate of digestion can be lowered by diluting the enzyme, thereby giving the experimenter more time to manipulate the apparatus and an opportunity to observe the effect of concentration on enzyme action.

"This effect can be observed in considerable detail if one performs the experiment on serial dilutions of the enzyme. The procedure requires a grid of 36 test tubes arranged in a pattern of six rows and six columns. Place one milliliter of water in each of the 36 test tubes. One milliliter of filtered saliva is then placed in each of the six test tubes that make up the first column (the first tube of each row). Swirl each tube gently to mix the solution. All tubes in the first column now hold two milliliters of solution. The remaining 30 tubes hold only one milliliter. Using a clean pipette, transfer one milliliter of solution from the first tube in the first row to the second tube in the first row. Swirl the second tube gently to mix the contents. Then transfer one milliliter of solution from the second tube to the third. Swirl. Repeat the procedure until the sixth tube of the first row contains two milliliters of solution. Remove and discard one milliliter of solution from the sixth tube. All tubes in the first row now contain one milliliter of solution. Repeat the procedure for each of the remaining five rows. All test tubes in the first column will then contain a 1:1 dilution of saliva-water solution. A11 tubes in each of the remaining five columns will contain dilutions in the ratios of 1:2, 1:4, 1:8, 1:16 and 1:32 respectively.

"The series of reactions and measurements is started by adding one milliliter of standard starch solution to the first test tube in the first column. Ten milliliters of indicator solution is added after an interval of 15 seconds, the activity is promptly measured and then tabulated, as in the initial experiment. Next, one unit of standard starch solution is added to the second tube of the first column (the first tube of the second row) and after an interval of 30 seconds is similarly measured and tabulated. Thereafter the identical procedure is repeated on all remaining specimens in the first column, the enzyme having been allowed to act prior to measurement for intervals of one, two, four and seven minutes respectively. The remaining five columns are then reacted, measured and tabulated on the same schedule.

"The tabulated data for each column is now converted to percentages of starch by reference to the calibration curve and plotted against time as shown in the accompanying illustration, a graph of the effect of concentration on enzyme action [above]. With these data at hand still other factors can be investigated that tend to modify the rate of starch conversion, such as temperature and acidity.

"The rate at which many chemical reactions proceed increases with temperature over a broad range of temperatures. Is this true of reactions that involve enzymes? Ptyalin might be expected to work most effectively at a temperature near 98 degrees F., the approximate temperature of the human mouth. To test the hypothesis, select from the graph just completed a concentration of ptyalin that produces, say, 80 per cent starch digestion in four minutes and prepare approximately 60 milliliters of ptyalin solution at this concentration as a stock specimen. Then improvise a constant temperature water bath by adding hot or cold water as required to a large beaker initially filled to about a third of its capacity with lukewarm water. Adjust the bath to 94 degrees F. Make up a series of starch dilutions from 100 per cent to O per cent as in the first calibration run. Place these dilutions in the water bath and, after they have reached 94 degrees, make a new calibration curve for starch at this temperature. Now place nine test tubes in the bath, each containing one milliliter of stock specimen, together with a single test tube containing 10 milliliters of standard starch solution.

"Keep an eye on the temperature of the bath and stir in enough hot water from time to time as required to maintain the specified temperature. When all test solutions have reached the temperature of the bath, transfer one milliliter of standard starch solution to each of the stock specimen solutions and, using the new calibration curve, carry out the procedure for measuring and plotting the rate of digestion. Thereafter increase the temperature of the bath in increments of two degrees to 110 degrees, repeating the complete procedure, including calibration, at each increment of temperature increase. If performed carefully, the experiment will demonstrate that the action of the enzyme increases to a maximum at a temperature somewhat above 98 degrees and thereafter declines to zero at 110 degrees. It is easy to demonstrate that the effect of high temperature on an enzyme is not reversible. A specimen of ptyalin first heated to 110 degrees shows no activity when subsequently tested at lower temperatures.

"Enzymes are similarly touchy with respect to the acidity or alkalinity of their environment. Normal saliva is slightly acid. On the pH scale universally used for expressing acidity and alkalinity, normal saliva has a value of pH 6.7. (A pH of 7 is neutral. Acid solutions have pH values lower than 7 and alkaline solutions values higher than 7. Each unit of the scale represents a tenfold change in hydrogen-ion concentration, a pH of 4 being 10 times more acid than pH 5 and 100 times more acid than a pH of 6. Similarly, a pH of 10 is 100 times more alkaline than a pH of 8. ) One might suppose that the reaction mediated by ptyalin would proceed most effectively in an environment of pH 6.7. The hypothesis can be tested by maintaining standard starch and ptyalin solutions at a constant concentration and temperature while varying the pH in small increments. The pH is altered by adding dilute sodium hydroxide (.1 molar) or dilute sulfuric acid (.1 normal) in small amounts to the specimen solutions and measuring the pH with Nitrazine paper, or by any comparable technique. Nitrazine paper, such as Squibb No. 5262, can be bought at drugstores.

"Adjust 8O milliliters of standard starch solution to pH 4 by adding dilute sulfuric acid drop by drop, and prepare a new calibration curve. Then, using dilute sulfuric acid, adjust one milliliter of ptyalin solution to pH 4, add the adjusted standard starch solution, after an interval of four minutes add 10 milliliters of indicator solution and carry out the procedure for plotting the digestion rate. Repeat this routine, including a new calibration run each time, in increments of two pH units from pH 4 to pH 10. If carefully performed, the experiment will demonstrate that ptyalin functions most effectively in a slightly acid environment.

"Many other factors are known to alter the rate of enzyme activity. The activity of ptyalin is increased by the presence of chloride ion in relatively low concentrations and by exposure to red and green light of moderate intensity. Its activity is destroyed, on the other hand, by the salts of heavy metals such as silver nitrate and mercuric chloride. These and still other environmental factors can be investigated by this same photometric procedure."

Seismographs of the type that employ pendulums swinging in the horizontal plane, such as those described in this department by A. E. Banks of Santa Barbara, Calif. [see "The Amateur Scientist," SCIENTIFIC AMERICAN, July, 1957], are subject to long-term drift. Gradual changes in the tilt of local terrain and in the dimensions of the instrument cause the center of the pendulum's swing to creep slowly toward one side of the instrument or the other and distort the record. The effect is of little consequence in the case of seismographs of the Galitzin type, which have a pendulum bob in the form of a coil of copper wire that swings in a magnetic field and translates earth movements as low-frequency electric currents. The voltage is proportional only to the velocity of the pendulum's swing and hence is unaffected by drift. But seismographs that employ a mechanical linkage, such as the Banks instrument, are another matter. In this arrangement a pivoted mirror is driven by the bob through a short lever arm of magnetized piano wire that is attracted to, and therefore held in contact with, an iron peg in the end of the bob. Any displacement of the bob, including drift, is communicated directly to the mirror system and appears as a bias in the recording. A nice solution of the problem has been suggested by J. P. Parker of the American Enka Corporation.

"Replace the magnetized piano wire with a coupling of viscous oil," he writes. "This can be accomplished by putting a shallow dish of oil on the seismometer arm and letting the oil drive a vane attached to the mirror lever [see illustration below]. A small restoring force must be applied to make the mirror seek the center of its angular excursion; this is achieved easily by fitting the mirror assembly with a weak centering spring."

Bibliography

SIMPLE TEST FOR THE APPROXIMATE ESTIMATION OF BLOOD CREATININE AND GLUCOSE IN ONE PROCEDURE. Emanuel E. Mandel and Edward B. Lehmann in The Journal of Laboratory and Clinical Medicine, Vol. 34, No. 5, pages 720-724; May, 1949.

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