NEPHELOMETRY IN THE STUDY OF PROTEASES AND NUCLEASES.

FIRST PAPER.

BY PHILIP ADOLPH KOBER.

(From the Harriman Research Laboratory, Roosevelt Hospital, New York, N. Y.)

(Received for publication, December 1, 1912.)

INTRODUCTION.

The author in studying ferments, required a method which would reveal, quickly and accurately, any change in the activity of the ferment during the course of an investigation. The follow- ing method, which is based on the use of a nephelometer, intro- duced into analytical chemistry by Richards,’ seems to meet those requirements.

The following four requisites can be given for a good method of estimating proteases ?

1. The substance used for a substrate should be a protein. 2. The protein should be soluble in the solution of the ferment,

in distinction to a reaction between phases. 3. Any and all unchanged protein should be completely precipi-

table by a convenient reagent. 4. This undigested protein should be determinable, accurately

and quickly. A suitable protein for peptic digestion is edestin,3 owing to its

solubility in weak hydrochloric acid solution. Casein is well adapted for tryptic digestion, owing to its solubility in weak alkali

1 T. W. Richards: Zeitschr. f. anorg. Chem., viii, p. 269, 1895; Richards and Wells: Amer. Chem. Journ., xxxi, p. ,235, 1904; Richards: ibid., XXXV, p. 510, 1906.

2 The subject of suitable units for proteases will be discussed in the near future.

3 Fuld and Levison: Biochem. Zeitschr., xi, p. 473, 1907. 4 Gross: Arch. f. ezp. Path , Iviii, p. 157, 1907.

485

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486 Nephelometry in the Study of Ferments

solution, and yeast nucleic acid in dilute solutions is similarly adapted for the study of nucleases.

Edestin5 is easily and quantitatively precipitated as edestan from its acid solution by 12 per cent sodium chIoride solution. Casein can be completely thrown down by a weak acetic acid solu- tion and other precipitants, while yeast nucleic acid can be ren- dered wholly insoluble by a slight excess of hydrochloric acid.

By using the specially devised nephelometer (easily made from the Duboscq calorimeter) described below, one can determine the amount of any undigested substance which is precipitated in the form of a suspension by these reagents, and thus follow the diges- tion of edestin with pepsin, of casein with erepsin and trypsin, etc., and yeast nucleic acid with nucleases quantitatively, with an error of less than 2 per cent. As a study of precipitants for casein and for yeast nucleic acid is still in progress the results on edestin only will be given in this paper.

METHOD.

(a> Description of th e instrument and its construction.

As the nephelometer described by Richards yields its best results only on taking a large number of readings (from ten to twenty),@j and since for ferment work so much time for readings is objectionable, an improvement was highly desirable.

A similar instrument of greater accuracy, one that yields reliable results with a few readings comparable to those obtained with a Duboscq calorimeter, would greatly enhance the value of the nephelometer for physiological chemists. No doubt the Richards instrument sufficed for the purpose for which Richards designed it and therefore efforts to improve it were heretofore superfluous. Since, however, the instrument is not desired now to yield a cor- rection to some other analytical process, but to form the basis of the analytical process itself and to yield all the figures of the determination, greater accuracy is required.

5 T. B. Osborne: Journ. Amer. Chem. SOL, xxiv, p. 28, 1902. 6 As stated in a private communication to the author, the Richards in-

strument rarely departs more than 4 per cent from the true value on a single reading; the mean of these readings is within 2 per cent; but to attain accuracy within 0.5 per cent, many readings are needful.

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Philip Adolph Kober

Without going into the various considerations connected with the improvement of the nephelometer, it is sufficient to say that the optical workmanship of the Duboscq calorimeter proved to be an excellent basis on which to build a nephelometer, and the following therefore represents an attempt to obtain greater accu- racy. 7

The change from calorimeter to nephelometer, and vice versa, can be made in a few minutes with the simple, easily made addi- tions described below. As most laboratories have a Duboscq calorimeter, an additional usefui instrument can be produced in this way with little expense.

The essential differences between the new and the older nephel- ometers are as follows:

1. In the new instrument, a plunger changes the height of liquid under observation.

2. A shutter supplements the action of the plunger in cutting off the amount of light.

3. The new nephelometer eliminates three possible sources of error: the meniscus, the indirect reflection of light from suspended matter in the lower part of the tubes, and the reflection of light from one tube to another.

4. The dark shade reduces any error due to the reflection of light on the eyepiece.

5. The new instrument is more adaptable to daylight. 6. Less liquid is needed for a test, 6 cc. being the maximum

amount needed. 7. The instrument is inclined, so as to make observations more

convenient, and to prevent air bubbles from forming when the plungers are first introduced into the liquid.

The accompanying drawing, for which I am indebted to Mr. Sugiura, shows clearly enough the arrangements without much further description.

The tubes containing the liquid can be made from soft glass having a bore of 15.2-0.3 mm. by anybody having a little skill in glass blowing.

The nephelometer shown here was made by the author, without any special tools or skill, from small packing-box boards

r Judging only from the readings with the new nephelometer, greater accuracy was obtained but as both instruments were not compared by the same person under similar circumstances, this statement needs further confirmation.

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488 Nephelometry in the Study of Ferments

FIGURE 1.

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Philip Adolph Kober

The essential points are: 1. The sides of the glass plungers are given several coats of black asphal-

turn paint, leaving the circular ends clean, with sharp and well-defined edges. This change can be made permanent, as it does not interfere with the use of the instrument as a calorimeter.

2. A removable wooden partition, fastened to a wooden bottom plate as shown in the drawing, is fitted to the instrument. This partition serves, not only as a guide for the two shutters, but prevents reflection of light from one tube to another.

3. The shutters which are held to the plunger brackets by a spring clamp, are made of the same material, and serve to eliminate stray reflections.

4. The tube holder, which is fastened below the metal base, by means of two rubber bands, contains two holes which receive the bottoms of the tubes.

5. The eyepiece is inclosed in a wooden or paper box, with an opening sufficiently large to admit a man’s head.

This eyepiece, as well as all other wooden additions, is covered with black asphaltum paint.

(b) General considerations.

As has been mentioned the instrument can be used with day- light, but for accurate work this light varies and is not always

sufficiently constant. Reliable results are obtained with a 100 watt “Mazda” lamp,8 placed, well-screened, about one-half meter from the nephelometer.

To use the instrument for quantitative work, it must be care- fully standardized with solutions of known strength, keeping the instrument under the exact lighting arrangements as in actual work. The amount of light observed in the eyepiece, as pointed out by Richards and by Wells is not proportional to the weight of the precipitate under observation, but seems to be dependent to some extent on the condition of the precipitate, the concentra- tion or amount of substance per cubic centimeter and the height and thickness of the liquid.

If a standard solution is always used in the same way, i.e., the same concentration and the same height of the liquid in the nephel- ometer, then the readings obtained with “unknown” solutions, plotted against the ratios of the solution, will follow with con- siderable accuracy, a definite and regular curve as may be seen in

8 I am indebted to Mr. W. F. Brady, Vice-President, New York Edison Company, for placing at my disposal a generous supply of different size tungsten lamps.

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490 Nephelometry in the Study of Ferments

Nephelometric Curvr

3OD

29.0

28.0

270

260

250

240

23.0

im

211)

20.0

19.0

18D

17.0

ml

15.0

figure 2. This curve, as may also be seen, is considerably lower than the “hypothetical” curve where the readings are inversely proportional to the concentration of the solutions. Richards and Wells assumed that when solutions of the same height gave the same amount of light, the ratios of the solutions were unity, and that around this ratio the “hypothetical” curve was correct. This is shown to be true by the curves in the chart within 1.5 per cent when the ratios are within 10 per cent of unity. It was therefore necessary, for Richards and Wells, to adjust thevolume of their solutions, until the ratios were within this limit.

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Philip Adolph Kober

In the following work with edestin a scheme is presented which practically eliminates this adjustment of volumes.

The curve in figure 2 can be expressed in the following equation.

y = z_ (I---z)K x 22

where y = height of “unknown” solution, x = ratio of solutions and s = height of standard solution.

By s is meant the reading of the nephelometer with the standard solution used as an “unknown,” or, in other words, when the ratio of the solutions is unity. This serves to eliminate any errors due to faulty light, tubes, plungers, etc.

Where K = a constant, obtained by substitution of standard- ization values.

Therefore, if once the value of K is derived for any given stand- ard solution and height of the standard solution, the nephelometric readings will give at once, upon calculation, the ratio of the solu- tions, and thus no further adjustments of volumes is necessary.

When the K obtained with one height of standard solution is compared with that of another height, it is found that K is pro- portional to the height of the standard solution, the equation then becomes for any heights of liquids within moderate limits.

YE-s- ~(1 - x)k

X 22

where k = 5 s

One has, therefore, the choice of using either one or more stand- ard curves and getting the ratios directly from a curve (as in figure 2), without calculation, or taking any suitable standard, adjusting either or both heights of liquids until the amount of light is matched, and obtaining the ratios by substitution in the nephelometric formula given above. A more convenient form when expressed in terms of y, is

x = s-y” +1/ (s + Sk)*- 4sky

2Y

g Of course, it is assumed that solutions which differ from a standard solution very much as may easily be observable macroscopically, will require, as in calorimetric work, a more suitable standard,

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492 Nephelometry in the Study of Ferments

when s is kept const’ant at 14.96 as in the curve, figure 2, and when K or NC has a value of 1.8 the equntion becomes

Ed&in ratios.

10 5-

RATIO 1* Y

14.9 14.9 15.1 15.1 14.9 ~-- 15.0 15.1

15.0 15.0 15.0

15.0 15.0 15.0

14.9 14.9

l-2.9 15.0

14.9 15.0 l-l.9 14.8

15 1 14.9

15 0 I.1 9 15.0

14.9 14.9 15.1

14.96

12 15 20 22.5 5 5 5 ~ 5

RTIO RlTIO RATIO ~ RATIO 0.882 0.750 0.600 0.545

i- Y N Y Y

1G 7 16.8

16.5 16.6

16.64 average

19.2 19 3 19.2

19.1 19.1 18.9 -- 19.0 18 9 19.0

19.0 19.1 19.2 18.9 19.3

23.1

23.0 22.9

23.4 23 0 22 6 22.8

22.8 23.1 22.7

23.2 23 2 23.0 23.3

23.0 22.9

24.9 24.G 25.0

24.83 tverage

25 30 5 5

FlATI RATIO 0.500 0.428

23 00 average

I I

Y

26.3 25.9 26.0

26.0 26.0 26.0 26.1

26.04 average

Y

28.8 29.5 28.7

28 5 28 7 28.6

28.5 29.0 28.8 29.0 -~ 28.81

average

*s = 15.0

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Philip Adolph Kober

The table on p. 492 gives the values obtained with edestin when 5 cc. of a 0.01 per cent solution, diluted with 10 cc. of sodium chlo- ride reagent, was used as a standard.

The numerator of the fraction indicates the number of cubic centimeters of the reagent which was used to dilute the protein solution: the denominator indicates the number of cubic centi- meters of 0.01 per cent edestin solution used. The third row of figures indicates the calculated ratios. The figures in columns are interlined to indicate the number of readings per solution. The average of all the figures of a column is given at the foot of each column, and is also indicated by crosses in figure 2. As may be seen, down to the ratio 0.50 they follow the curve quite satisfactorily, but beyond this ratio they deviate slightly, owing to a factor that is produced by a difference in the height of the liquids. In actual work this may easily be eliminated by following t,he curve of observed readings, or by choosing a more comparable standard. It may also be taken into account in calculating the ratios, as illus- trated in the following paragraphs.

This factor that causes t’he nephelomet.ric readings to be slightly too low, especially when the ratios are below 0.500 and when the per cent of edestin of any of the solutions is below 0.0033 per cent, is due t,o the fact that even pure water will produce some light in the eyepiece. This arises, no doubt, from imperfections of the apparatus, it being very difficult to exclude all stray reflection of light. The equivalent of this amount of light is esti- mated by making a control determination on pure water, using a 0.0005 per cent solution of edestin as a standard. Under the conditions given in this paper, 28.0 mm. of pure water matched a 0.0005 per cent solution of edestin at 5.4, 4.7, 4.5, 4.8, and 5.7 mm. = 5.0 mm. average. Assuming this proportion to hold for all heights of distilled water, we find that 23 mm. of water will give the same amount of light as 5.0 mm. of 0.0005 per cent edestin (excluding the amount of light from its 5 mm. of water; thus 28.0 - 5.0 = 23.0 mm.) and therefore 1.0 mm. of water = 0.21 mm. of 0.0005 per cent edestin. Hence, when very weak solutions are determined, care should be used to see that control corrections are applied, or eliminated by careful standardization.

In order to test the general applicability of the nephelometric formula given above, other heights of solutions and ratios than those covered in the curve were used as is shown in the table below:

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494 Nephelometry in the Study of Ferments

3. % i"

p$' Q+a $2." iz+ H il

D tl

II H

0.489 0.477

0.504 0.493 0.495

(l-3) N:sCi I 0.01 per 0.01 m?r

rent. (l-3) NaCl cent 1 edestin ~ edestin

I I

15.1 26 9 10.0 18.2

14.8 25.9 9 9 17.6

~-- I --- 0.500~ 0.56: 0.500 0.54:

0.500 0 571 0.500 0.56: 0.500 0.571

cc. cc. cc. IO 3 i 2+5

10 5 2.5

8.0 14.0

0.5001 0.563; 0.494 0.500~ 0.500'

0.550 0.488 0.568 0.500

' 0.5001 0.554 I 0.4i2 0.500 0.5681 0.4S9

10 5 ( 25 15.1 26.8 12.0 21 8 17.0 29.9

15.2 27.5 15.2 26.8

0.491 ,500 1.562. Averrtgc.

j cor- , rected

Y

25 5 45 5 / 28.9 13 0 32.3 3 300~ 0.450 0.2iTi I 11.1 3.0 12.3 3.3001 0.451 0.284

10.0 24.G 13.0 31.7

3.300 0.4501 0.282 3.3OC 0.4561 0.289

5.2 28.0 4.8 28.0

Avmgc.. 0.282

These results are sufficient to show that by the use of either the formuia within moderate limit’s or curves prepared from three or four sets of readings, one can use the nephelometer for the determination of suspended protein and organic substances with the accuracy and with the ease with which the best calorimetric work is done.

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Philip Adolph Kober 495

Since this nephelometric formula seems to hold for a protein of high molecular weight, it was of interest to try its application to argentic chloride suspensions. In order to avoid the corrections necessary when very weak solutions are compared, as indicated above with edestin, relatively strong solutions were used.

0.00572 per cent

AgCl

0.00572 per cent

A&l Hz0 Hz0

‘-

cc.

10

cc.

10

30

cc.

10

10

10

15.0 15.1 15.0

15.0

23.8 23.8 23.6 23.5

23.7

1.00

0.5oc

0.5oc

1.00 1.00

3.63: 0.500

D.628 0.491

1.00 i.oa

0.631 0.509

0.001432 per cent

AgCl

10

10

0.001432 percent ~

A&l

15.0 23.6 23.4

' 23.5 ! 23.5

23.5

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496 Nephelometry in the Study of Ferments

As may be seen from the results with argentic chloride the nephelometric formula holds for considerable variations in con- centrations; the strongest solution being eight times as strong as the weakest, or a ratio of 0.125. It is probable that when cor- rections are made, especially for the weaker solutions, the result will be still more consistent with the formula. It is to be noted that these experiments give kAgor = 0.21 and /cEDESTIN = 0.12.

(c) Solutions and reagents.

Edestin solutions were made as follows: 1. Stock solution: 0.1000 gram of Merck’s edestin, weighed

accurately in a 50 cc. beaker, was moistened with sufficient water to make it possible to rub the substance into a paste. After thor- ough rubbing, 3 cc. of & hydrochloric acid was added which dis- solved all but a trace of the suspended substance. After allowing the solution to settle for an hour, it was filtered through a well- washed filter paper into a clean, 100 cc. graduated flask. This was done to prevent foreign particles and fibers of filter-paper from getting into the solutions. On bringing the solution up to the mark, adding a few drops of chloroform, shaking and allowing it to stand for twenty-four hours, it was ready for use.

2. Standard solution: 10 cc. of this stock solution were diluted to 100 cc., thus making it a 0.01 per cent solution. One volume of this standard solution, with one, two or more volumes of l-3 NaCl solution gave suitable standards for nephelometric work. These standards will remain as suspensions one or more hours depending on the dilution. In the work given above a fresh stand- ard was made for every determination.

3. Precipitant for acid edestin or edestan: (l-3) NaCl solution. a. Stock solution: A saturated solution of sodium chloride. b. Reagent. (1-S) NaCZ: One volume (250 cc.) of saturated NaCl

solution, with three volumes of water (750 cc.) was found to give satisfactory results in precipitating the edestan completely.

(d) Directions.

For precipitating substances. Thus far, satisfactory results have been obtained by adding the precipitant drop by drop from a carefully standardized burette to the standard solutions contained

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Philip Adolph Kober

in a beaker or small Erlenmeyer flask. Test tubes should be avoided as it is difficult to shake the solution properly, without adding a stopper or introducing minute air-bubbles into the solu- tion. An Erlenmeyer flask or beaker shaken in a rotatory fashion gently is most satisfactory.

The tubes may be cleaned and dried with alcohol and ether, or any other suitable way so long as no dust or lint is thereby intro- duced.

For reading the instrument: The plunger, being painted with asphaltum paint, obviously cannot be cleaned and dried with alcohol and ether; but may be rinsed with some of the liquid to be examined, or may be wiped carefully with a lint-free cloth or lens paper. To use the instrument, it is well to insure proper working conditions by putting into both tubes some standard liquid and comparing the heights. If the readings are consistent and practically equal on both sides, one may presume the light- ing arrangements and other parts are in proper adjustment.

The following suggestions were helpful in obtaining good results. 1. The instrument was kept in a separate room, where all the

lights could be extinguished at pleasure. 2. After the tubes containing the suspensions were in position,

the room was darkened completely for a few minutes, to rest the eyes. Using the instrument directly after coming from a well- lighted room produced inferior results.

3. Both eyes were kept open in reading the instrument. This was easily accomplished with the shaded eyepiece, and produced less strain.

4. It was found advisable not to make the adjustments too rap- idly or too constantly in order to avoid inaccuracies due to eye- strain. The final adjustment was made only after relaxing the eyes for a few minutes. The eyes were used alternately, the ap- proximate adjustment was made with one eye and the final always with the other.

5. When much nephelometric work was done necessitating going t.o and from a strongly lighted room, often, smoked glasses were found restful and advantageous.

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498 Nephelometry in the Study of Ferments

SUMMARY.

I. A micro-chemical method for following the digestion of a soluble protein, edestin, based on the use of a nephelometer is given.

II. A new and sensitive nephelometer, easily made from and into a Duboscq calorimeter is described.

III. The readings of the nephelometer plotted against the ratios of the solutions, for a given standard solution and a given height of standard, seem to follow a uniform curve which can be expressed

s(l-dk in the equation y = t - 22 where y = height of (‘unknown”

solution, s = height of standard solution, x: = ratio of solutions. The studies of various precipitants for protein and other organic

substances in dilute solutions are in progress with the view of extending the application of this method generally. The ease, the rapidity and the accuracy of the method would make it very use- ful, if the proper precipitants can be found. By proper dilution it can be used for large amounts of substances, and is sensitive enough to determine 0.00002 gram with a percentage error of less than 2 per cent. The determination of casein in milk and the estimation of minute quantities of ricin are receiving immediate a.ttention. by guest on A

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Philip Adolph KoberPAPER

PROTEASES AND NUCLEASES: FIRST NEPHELOMETRY IN THE STUDY OF

1913, 13:485-498.J. Biol. Chem. 

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