METHODS and APPLICATIONS of ENZYME STUDIES in HISTOLOGICAL CHEMISTRY by the LINDERSTROMLANG HOLTER TECHNIC* DAVID GLICK Research Laboratories of the Mount Zion Hospital, San Francisco, California
T
HE STUDY of enzymes as a means of contributing to the understanding of life processes has been retarded largely because of practical difficulties. For instance, an enzymatic investigation of microorganisms can give only an average value of their activities, since enzyme extracts must be prepared from large numbers of the organisms, which exist in many different stages of development, making it impossible to learn much about the single organism. Then, too, an enzymatic study of tissue is confused because the tissue contains dierent types of cells with different functions. Accordingly, with the present macro methods, an enzyme extract of tissue can give only a ronglomerate picture of the enzyme status in the cells. Of course, in the ideal case, enzyme investigations would be carried out on single cells, but as LinderstromLang and Holter showed, this would require a 100,000fold refinement of current macro methods. As an approach to this ideal state, Linderstrom-Lang and Holter, a t the Carlsberg Laboratory in Copenhagen, have devised an ingenious apparatus which they and their collaborators have adapted to micro enzyme investigations. These methods refine the usual macro procedures 1000 times without loss of,accuracy, and even make possible enzyme or chemical studies on single microtome slices of tissue or biological %nits such as a sea-urchin egg. By these means, a small group of cells of the same kind, or of the same location, can be studied enzymatically, and by means of parallel microscopic study, the enzyme functions of definite cells can be determined.
capillary, with a slight bend a t the-tip so that it can make contact with the wall of the reaction vessel. The pipet is calibrated by etching a mark a t the proper position on the stem. The vessel with the liquid to be pipetted is placed upon the table and raised until the tip of the pipet dips under the surface of the liquid. Then stopcock H is closed, K opened, and a gentle suction applied through tube S until the liquid is drawn just above the mark on the stem, which is observed through the low-power microscope, M. K is then The reaction vessel consists of a small tube 2 cm. closed, and the moment the meniscus falls to the mark, long with a capacity of about 250 cmm. (A, Figure 1). the table is quickly lowered to clear the pipet of the It is held upright by inserting the closed end into a liquid. The surface tension a t the tip of the pipet is small wooden block (1). sufficientto prevent the liquid from running out. Now There are two types of pipet employed (1). The the vessel to receive the liquid is placed upon the one (Figure 1) consists of a glass tube drawn out to a table and raised until the tip of the pipet touches the * Contribution to the symposium on The Chemistry of Eu- wall near the bottom; then the liquid is forced out zymes conducted by the Division of Agricultural and F w d Chem- under constant pressure by opening H. The pressure istry at the eighty-eighth meeting of the American Chemical is determined by the height of the column of water beSociety, Cleveland, Ohio, Sept. 12, 1934. 7 Available from the Burrell Technical Supply Co., Fifth tween the surface and the open end of the glass tube in T. Ave., Pittsburgh, Pa.
While this pipet can be used for small amounts of liquid (5 to 10 crnm.), the other is an automatic type to be used to simplify the accurate measurement of larger amounts (20 to 40 crnm.) (Figure 2). It con-
sists of a narrow glass tube drawn out to a capillary at both ends so that, with one atmosphere pressure, the fineness of the tips will prevent the liquid from running out. R is the siphon arm connecting the pipet to the reservoir of liquid placed about 50 cm. above the instrument. The pipet is filled in the following .manner: L is closed, K and H are opened so that the liquid fills the outer chamber. When the level is a few mm. above the upper tip of the inside pipet, H i s closed, and the pressure from the reservoir forces the liquid into the pipet. Then with K closed, L and H are opened to drain away the excess liquid. The pipet is emptied by closing L and H a n d opening K. The reservoir pressure compresses the air in the chamber, and forces the liquid out of the pipet. A pipet of the first type with a capacity of 7 cmm. operated with an error less than 0.3%, while the latter pipet of 30 cmm. capacity has an q r of measurement of less than 0.1%. The method of stirring ( I ) the very small volumes of liquid used in this technic consists in using little sealed glass balls which have been filled with iron powder. One of these so-called "fleas" is placed in the reaction tube and stirring is effected by means of an electromagnet E (Figure 3) which is connected in series with an interrupter. The magnet goes off and on about two times a second causing the "flea" to jump up and down, thus giving very a c i e n t stirring. Stoppers are made fat the reaction vessels by closing one end of a short section of rubber tubing with a glass plug. When it is desired to keep a digestion mixture a t a given temperature, the stopper is placed on the reaction tube, and the whole is immersed in the water of a thermostat by a copper wire twisted around the tube at one end, and suspended by the other from a rod above the thermostat. The micro buret (Figure 3) is made of a capillary
-1
tube about 60 cm. long. Its total capacity is 100 cmm. graduated in 0.2 cmm. Readings are taken to 0.02 cmm. In the bottom of the buret is a mercury reservoir, and turning in the screw forces the thread of mercury up the tube which, in turn, forces the liquid out of the buret. Readings are mad6 by noting the position of the top of the mercury column. The buret is filled by reversing the screw while the tip of the buret dips into the standard solution. The tip of the buret, S,dips into the liquid to be titrated and a "flea" stirrer agitates the solution in the vessel during the titration. The endpoint is determined by :omparing the color of the titrated liquid with that of a standard color liquid in another tube placed beside it. Evaporation is reduced during the titration by the loosely fitting glass cap around the tip of the buret, held suspended by two threads. The titration can he duplicated to 0.020.04 cmm. METHODS
With the apparatus just described, LinderstromLang .and Halter devised a method for the determination of proteinase and peptidase activity ( 2 ) . Seven cmm. of enzyme preparation (usually a 30% glycerol extract) is added to 7 cmm. of buffered substrate (the
JUNE, 1935 bufferina is effected merely by addina suitable amounts of acid or base to the proteinbr amino acid used as substrate). The whole is then digested a t 40°C. for two hours, and the increase in NH2 groups is determined by the Linderstrom-Lang acetone titration. One hundred fifty cmm. acetone containing naphthyl red indicator is added to the reaction mixture, and the titration is carried out with N/20 alcoholic HC1. The maximum deviation encountered in a series of these titrations was 0.08 cmm. (an error of 0.08 cmm. N/20 HCl equals 0.056 gamma amino nitrogen). Linderstrom-Lang and Holter have developed a method for the micro determination of sugars which could be adapted to carbohydrate studies (5). It is essentially a Willstiitter-Shudel iodometric titration. Seven cmm. of sugar solution of a concentration up to is pipetted into the bottom of a reaction vessel which has been paraffined internally. A "flea" (D, Figure 4) is added, 50 cmm. of carbonate buffer
This buret is the same as the one previously described except that there is an air space between the mercury and the standard solution. This bnret must be used with solutions that might be influenced by contact with mercury. Turning screw S e6ables either a small positive or negative pressure to be applied over the left column by raising or lowering the right column. Thus the liquid can be drawn into or ejected from the bnret. This method was checked for the titration of glucose, maltose, sucrose, fructose, and starch separately, as well as mixtures of these. Last year Linderstrom-Lang and Holter devised a micro method for the determination of ammonia (6) with the view of applying it to micro enzyme investigations. It may be used for urease determinations, and in a recent communication the author was informed that it has been adapted for Kjeldahl analyses. The (pH 10.6) is added, and the two solutions are mixed. solution to be analyzed for ammonia, up to a volume of Then 14 cmm. of 0.15 N IZin K I is introduced from 14 cmm., is pipetted into the bottom of a paraffined the automatic pipet so that the Izsinks to the bottom reaction vessel. A "flea" is added and with the tube of the drop. Then 50 cmm. 1.2 N HzS04is introduced horizontal, a drop of 2 N NaOH is placed near the with a hand pipet (Figure 5) to form a droplet, B, above bottom, but not touching the ammonia solution. A the mixture, C. b o t h e r film of liquid A consisting of film of water (35 cmm.) is placed near the top of the 30 cmm. 0.3% starch solution in N/100 HzS04, is vessel. The tube is now placed in a vertical position placed above B. These films prevent evaporation of which causes the drop of base to run down into the Iz. After 30 min. the reaction tube is placed inside a ammonia solution. Immediately, 7 cmm. of standard centrifuge tube and centrifuged for 1 min. a t 1500 HCl containing hromcresol purple is pipetted into the R.P.M. The centrifuging causes the liquid films to water film. The vessel is closed with a stopper, by collapse and mix with the alkaline mixture a t the bot- means of a "flea" the unknown is mixed with the NaOH, tom. After centrifuging, the excess Iz is titrated with and the vessel is half immersed in a thermostat a t 40 deN/20 N~LIS~OS using a second type of buret (Figure 6). grees for 5 hours. This is more than enough time for
all the ammonia to distil out of the lower drop into the upper drop containing the standard acid. The excess standard acid is now titrated with N/100 sodium borate. The titration arrangement is s h o w in Figure 7. The tubes are placed diagonally so that the "flea" will have the loncest path in which to travel. The color . possible . of the endpoint is determined by comparison with a similar film buffered to pH 5.9 in an adjacent tube. The buret is of the first type except that it is connected to a bottle of standard borate through B. The table movable in 3 dimensions by the three screws is the type of titration table now employed for all work. The greatest deviation between single estimations is 0.1cmm. N/100 borate (equal to 0.014 gamma nitrogen). The error, taking average values, is of course much less. Working with Linderstrom-Lang and Holter, the author adapted the apparatus to the micro estimation of lipolytic enzymes (8). Titration of the acid liberated by enzymatic hydrolysis of fat or ester could not be determined bv titration with alkali since COz of the air interfered. Accordingly an acidimetric method was developed with an endpoint below pH 7, The buffer employed was a solution of pH 8.7 composed of 0.1 N
NaOH with glycine to 0.4 N. The pH of glycine is about 9.5,and that of butyric and other organic acids present in the usual substrates is near 4.5. Hence the pH for a sharp endpoint was chosen a t 6.5,bromthymol blue being used as the indicator. The acid formed by the enzyme action reacts with some of the base of the buffer so that the amount of standard acid needed to reduce the pH to 6.5 is less than that required in a control tube with zero enzyme action. The determination is carried out by adding 6 cmm. 30% glycerol extract of the enzyme to 7 cmm. buffer-substrate mixture. The solutions are stirred with a "flea," incubated a t 40 degrees, the enzyme adion stopped by adding 50 a m . 2% phenol solution containing bromthymol blue, and the whole is titrated with N/20 aqueous HCI. The greatest deviation in a series of titrations was 0.14 cmm. That the micro technic might be applied to the determination of phosphatase, the author with Linderstrom-Lang and Holter developed a micro method for inorganic phosphate depending upon the precipitation of MgNH4P04. The precipitate formed in a small reaction tube is washed by centrifuging down the precipitate, decanting the supernatant liquid by withdrawing it with a capillary tube, adding a washing liquid (alcohol), stirring, again centrifuging, decanting, and washing three times with acetone. The precipitate is then dissolved in N/10 HC1 and the excess HC1 is titrated in a manner that will not include the HsPO, formed by solution of the MgNH4P04 with HCl. Fortunately, H3P04is not ionized in acetone, so that an acetone titration with base will measure only the excess HC1. Because of carbonate formation and the fact that alcoholic KOH does not flow smoothly through the fine capillary of the buret, theus?of alkali for the titration of the HC1 was abandoned. Instead the acetate ion was used. Since acetic acid formed by the neutralization of acetate ion and hydrogen ion is not ionized in acetone, the acetate ion will react with any hydrogen ions present with the same ease as hydroxyl ions would. Hence the standard solution >employed was M/20 CH3CO0 NH4 with added CH8COOH to stabilize the solution. By this method 5 gamma of phosphorus may be determined with less than 1.0% error, and 1 gamma with about 5.5% error.* It might be mentioned that the author has succeeded in developing the micro titration of vitamin C with 2,6-dichlorophenol-indophenol(15). The titrations are reproducible to *0.0001 mg. of ascorbic acid. This method is now being applied to a histochemical study of the adrenal and other organs. APPLICATIONS
The applications of the technic deicribed have been very diverse. First, Linderstrom-Lang and Holter, because they were interested in the relation between enzyme activity and growth of living organisms, iu-
* In a recent communication to the author. Linderstr6m-Lang and Holter state that they have been able to apply the micro technic to the determination of phosphorus by the Lahmann method.
vestigated the distribution of peptidase in the roots and sprouts of sprouting barley ( 2 ) . From the sprout or root, microtome sections 2 0 0 ~thick were taken along the longitudinal axis, and the peptidase activity of each section was determined. I t is seen that the maximum activity is found just behind the growth center V (Figure 8) which is situated under the root cap. Because of the changes in the ratio of the splitting of the alanyl glycine and leucyl glycine the authors conclude that there must be either two peptidases, or one enzyme and one or two activators or inhibitors.
which were then extracted with 60% glycerol. The extract was allowed to act upon edestin and micro titration was performed to determine the extent of the hydrolysis. Similarly the proteinases of the hairs and leaf tissue were studied. About 500 hairs were shaved from a leaf, and crushed with 300 cmm. of glycerol to form an extract. Leaf tissue extract was made by extracting 7 leaves, with the hairs shaved off, with cc. of 60% glycerol. After centrifuging these preparations, 7 cmm. of extract was used for each determination. The pH determination was carried out with a micro quinhydrone electrode devised by Kuntara (14). Acid was added to the edestin substrate to form the properly buffered solution. The results shown in Figure 9 reveal a proteinase in the secretion with a pH optimum of 3.2 (curves I and 11) and an endoenzyme in the tissue of pH optimum a t 4.3 (curve IV). Curve I11 is that for the hairs. and it shows an over-
DISTANCE FROM ROOT TIP IN mm.
2
I
l I
I 2
I 3
l
,
I
_
1
4 S 6 DISTANCE FROM LEAF TIP IN mm.
L 8
I n the next application of their technic, Halter and Linderstrom-Lang studied the proteinases of Drosera rotund$olia, an insectivorous plant (3). They were interested in the enzymes this plant secretes to digest for its food the insects that fly into its trap. The leaves forming the trap are lmed with fine red hairs, and tiny drops of secretion appear on them. This secretion was transferred to pieces of filter paper (2 X 2 mm.)
lapping of the properties of the enzyme of the secretion and of the tissue. The next application of the micro technic was a study of the peptidase content of the eggs of the two marine invertebrates, the California sand dollar, and Urechis caupo (4). The eggs of these animals weigh approximately gamma wet weight or about gamma dry weight. The Urechis caupo egg can hydrolyze its own weight of alanyl glycine in one hour a t 40°,while the California sand dollar egg hydrolyzes twice its weight of the substrate in one hour a t 40'. An egg in sea water was extracted with 7 cmm. of glycerol, 7 cmm. of an alanyl glycine solution containing NaOH to bring the pH to 7.5 was added, and the digestion and titration were carried out as described previously for peptidase determinations.
The yolk portion is in the lower part, and so has greater activity per unit weight than the rest of the egg.
A
A
C
1
4
4
1.32. 1.20 1.16, 1.27 1.30, 1.32 5.01.4.97 5.05. 5.03 4.89. 4.93 4.91. 5.05
/
5.05. 5.07 4.87.4.91 4.88.4.96
1
5.32. 5.22 5.18, 5.28
1
1.26
P i n t day
4.M
p i n t day
4'99 4.92
Second day
3 days later
5'25
Table 1 shows the order of magnitude of the hydrolysis. It also shows that eggs from the same animal have nearly the same enzyme activity, though this activity varies from animal to animal. Table 2 shows that after fertilization the enzyme activity first falls, then rises to a maximum, and finally falls again. By cutting the eggs with a glass needle on a micro manipulator it was shown that the part containing the nucleus seems to be a little more active than the other part, but. the sum of their separate activities equaled that of the whole egg. It was shown also that the sperm of these animals, and the jelly surrounding the California sand dollar, are without peptidase activity.
C.
8 hrs.
E. 4 h n .
Before fertilization 0.25 3 6.3 32
3.27 ,253 3.06 3.65 1.74
Before fertilization
1.18 0.92 0.93
0.75 1.4
*'.
06j.ird Intact Egg Upper Part Lower Part Intact Egg upper Part Low- Part
Another application of the histological micro method involved the study of the distribution of the enzymes in the stomach and duodenum (9-13). Fresh hog stomach and duodenum kept a t -lZ°C. until needed, was the source of the material. A small sharp cork borer of 2 mm. diameter was used to drill out, through the wall of the organ, cylinders of tissue which were placed on a frozen section microtome a t once and sectioned. In different experiments the slices taken were from 10 to 250 thick. Each slice was placed in a separate reaction vessel, 7 cmm. 30%
ODENUM
4 divisions of egg First movements of embryo Large, well developed motile ernbrvoo
1 division of ex.
Working with Linderstrom-Lang and Halter, Philipson studied the eggs of Psammechinus miliaris (7). The eggs were placed between a layer of isotonic sucrose solution and a less dense layer of sea water. Upon centrifugation a t from 8000 to 13,000 R.P.M. the eggs divided into two or more parts with different specific gravities, appearance, and transparency. Table 3 shows that the sum of the peptidase activities (using 3 eggs and alanyl glycine as substrate) of the parts is equal to that of the intact egg. The upper and lower parts have about the same enzyme activity though the volume ofthe upper portion is 4 times that of the lower.
DISTANCE FROM SURFACE IN mm.
0
05
lo
I5
20
2s
I 1 % CISTANCE FROM SURFACE IN mm.
FIGURE 10
30 I
glycerol was added to extract the enzyme; 1 to 2 hours later, 7 cmm. substrate was added, and the determination was carried out as already described. Besides the specimen taken for enzyme estimation, a similar cylinder of tissue was removed for histological study. The results of this work are shown in Figure 10. As an example, a section through a gland in the fundus of the stomach is pictured. The epithelial cells (A) lining the inside of the stomach soon give way to the parietal (B) cells, which gradually disappear farther down from the surface until the chief (C) cells predominate. Below the C cells is the muscular layer composing the outside of the stomach pouch. Now it may be seen that the HCl has a maximum concentration about '/z mm. from the inside surface, corresponding exactly to the presence of the B cells. Hence, here is direct evidence that the HCl secretion is connected with the B cells. The peaks in the pepsin and peptidase curves correspond to the maximum incidence of C cells, thus fixing the responsibility for the presence of these enzymes upon this particular type of cell. It should be noted that the fundus is the only portion of the stomach where any acid is contained in the cells. It might be mentioned that the acid was ,determined by titration with N/100 borate in a manner similar to that described in the ammonia method. The relatively high concentration of pepsin in the fundus, and low concentra-
tion in the pylorus and cardia, is just reversed in the case of peptidase. An interesting feature of the results obtained with duodenum is the dip in the curves about 11/2 mm. from the inside surface. At this point a thin muscle layer separates the surface cells on the inner side from the Brunner glands on the other side; below these Bmnner glands is the heavy muscular layer. The dip is obviously due to the weaker enzyme activity of the muscle tissue. The lipase curves show a consistent peak just under the inner surface of the stomach and duodenum, and a fairly high activity in the muscular layer comprising the outer wall. This then should give an idea of the apparatus, methods, and applications of the Linderstrom-Lang Holter technic from its beginnings in 1931 to the present. The value of these methods for further enzyme work, and for purely chemical investigations of a micro order, needs no comment. By extending the view of those working in the life sciences beyond the field of the microscope into the chemical constituents and actions of individual cells, the Linderstrom-Lang Holter technic has indeed marked a real step forward. The author wishes to express his sincere gratitude to Drs. K. Linderstrom-Lang and Heinz Holter of the Carlsberg Laboratory, Copenhagen, for theirsuggestions in the course of preparation of this manuscript.
LITERATURE CITED
K. LINDERSTR~#M-LANO HEINZHOLTER,"The estimation of small cleavages caused by enzymes," Comfit. rend. trou. bboratoire Carlsberg, 19, No. 4 (1931); Z. physwl. Chem., 201, 9 (1931). K. LINDER~TR$M-LANO AND Hsmz HOLTEE,"The distribution of peptidase in the roots and sprouts of malt," Compt. rend. traw. laboratoire Carlsberg, 19, No. 6 (1932); Z. physiol. Chem., 204.15 (1932). HEINZ HOLTERUND K. LINDERSTR#M-LANO, "iiber die Proteinasen von Drosera rotundifolia," Z. Physiol. Chem., 219,223 (1932). K. LINDERSTR@~-LANG, "The peptidase Content of Some marine invertebrates," Compt. r&d. trev. laboratoire Carlrberg, 19, No. 13 (1933); Z. physiol. $hem., 215, 167 11022)
K. LINDERSTR#M-LANG AND HEINZ HOLTER,"A micro. method for the estimation of sugars." Compt. rend. trau. labratoire Carlsber~. e. 19. ,No. 14 (1933). , , K. LINDERSTR$M-LANO AND HEINZ HOLTER,"A micromethod far the estimation of ammonia," ibid.. 19, No. 20 (1933); Z. physiol. Chem., 220,5 (1933). TOREPRnlPSON. "A. The peptidase in the eggs of Psammechinus miliaris. B. The distribution of mitachondria in centrifuged Prammechinus eggs," Compt. rend. trav. laboraloire Carlsberg, 20, No. 4 (1934); Z. physiol. Chem.. 223.119 (1934). GLICK,"A micro method far the determination of
DA&
lipolytic enzyme activity," Compt. rend. Ira". labomtoire Carlsberg. 20, No. 5 (1934); Z. physiol. Chem., 223, 252 (19x4). ,----, HEINZHOLTERAND K. LINDERS~~#M-LANG. "The distribution of pepsin in the mucous membane of hog stomach," Compt. rend. tmu. kzboratoire 'Carlsberg, in press: Z . physiol. Chem., 226, 149 (1934). K. LINDERsTRplM-LANGAND HEINZHOLTEE,"The distribution of acid in the mucous membrane of bog stomach," Compt. rend. trev. laboratoire Cerlsberg, in press; Z physiol. Chem., 226, 173 (1934). K. LINDERSTR#M-LANO AND HEINZHQLTEE,"Distribution of DeDtidase in mucous membrane of stomach and duodenum of hog," Compt. rend. trau. labratoire Curlsberg, in press; Z . physiol. Chem., 226, 177 (J934). DAVIDGLICK,"Distribution of esterase m the mucous membrane of stomach and duodenum of hog," Compt. rend. trau. kzboratoire Carkberg, in p r w ; Z . physiol. Chem., 226,186 (1934). K. LlNDERSTR$h-LANG. HEINZ HOLTER,AND A. S~EBORG OHLSEN,"The enzyme distribution in hog stomach as a function of its histological structure," Compt. rend. trau. laboratoire Carlsberg, in press; Z . physiol. Chem., 227. 1 I192A) W. K ~ A R "EineneueMikrodoppelchinhydronclektrode," A , Z . physiol. C h . , 204, 54 (1932). DAVIDGLICX, "The chemical determination of minute quantities of vitamin C,"J. B k l . Chem., in press.
