H[arry] Gideon Wells.

Chemical pathology; online

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applied cells, and this substance seems to be related to mucin.
It can generally be brought out by staining with silver nitrate,
and Macallum ! points out that this reaction is merely a micro-
chemical test for chlorides, and indicates that the cement sub-
stance contains them in larger proportion than does the cyto-
plasm.

1 Proceedings of the Royal Society, 1905 (76), 217.



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CHAPTER II
ENZYMES

Every cell is constantly accomplishing an enormous number
of chemical reactions of varied natures, at one and the same time ;
how many we do not know, but the score or more that we do
know to be constantly going on in the liver cell, for example, are
probably but a part of the whole. Furthermore, reactions take
place between substances that show no inclination to affect each
other outside the body, and proceed in directions that we find it
difficult to make them take in the laboratory. Sugar is being
constantly oxidized into carbon dioxide and water, a decomposi-
tion that requires high degrees of heat or powerful chemicals to
bring about in the reagent glass. Proteids are being continu-
ally broken down into urea, carbon dioxide, and water ; yet to
split proteids even as far as the amino-acid stage requires pro-
longed action of concentrated acids or alkalies, or superheated
steam under great pressure. 1 But all the time in the cell a
multitude of equally difficult changes is going on at once,
within its tiny mass, always keeping the resulting heat within
a fraction of a degree of constant, and the resulting products
within narrow limits of concentration. We have already indi-
cated the means used to keep the concentration of the cell prod-
ucts within safe limits ; namely, the processes of diffusion and
osmosis and their modification by the cell structure. The
forces that bring about the chemical reactions reside, we say, in
enzymes, although in so doing we only shift the attribute
formerly conceded to the cell, to certain constituents of the cell
whose nature and manner of action are equally unknown.
When the only enzymes that were known were limited to those
secreted from the cell, and found free in fluids, such as pepsin
and trypsin, the chemical changes that went on in the cell were
ascribed to its "vital activity." Buchner, by devising a
method ^ crus h y^^ ceU^ and finding the exposed cell con-
t* nt8 able to produce the same changes in carbohydrates that
» °^)* themselves did, proved the existence vrithm living



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62 ENZYMES

substantiated the belief of their existence that had become
general before it was thus finally corroborated. Growing out
from this and subsequent experiments has come a larger and
larger amount of evidence that many of the chemical activities
of the cells are due to the enzymes they contain, until now the
point is reached where one may rightfully ask if cell life is not
entirely a matter of enzyme activity. There are certain facts,
however, which seem to indicate that there are some essential
differences between cells and enzymes. One of the most
important of these is the difference in the susceptibility to poi-
sons of enzymes and cells. Strengths of antiseptics that will
either destroy or inhibit the action of living cells, such as alco-
hol, ether, salicylic acid, thymol, chloroform, tuluol, and sodium
flouride, will harm free enzymes in solution little or not at all.
This fact has been of great assistance in distinguishing between
the action of enzymes and of possible contaminating bacteria in
experimental work. Although this difference between enzymes
and cells is characteristic, it does not finally decide that the cell
actions are not enzyme actions, for it may well be that the poi-
sons act chiefly by altering the physical conditions of the cell
so that diffusion is interfered with, thus seriously interfering
with the exchange of splitting products between different parts
of the cell, and checking intracellular enzyme action, which we
shall see later requires free diffusion of the products for its con-
tinuance. 1 At the very least, however, we may look upon the
intracellular enzymes as the most important known agents of
cell metabolism, and consequently of all life manifestations,
and the changes they undergo or produce in pathological condi-
tions must be fully as fundamentally important as is their rela-
tion to physiological processes. It therefore becomes necessary
for us to consider carefully —

THE NATURE OF ENZYMES AND THEIR ACTIONS 2

Since up to the present time no ferment has been isolated in
an absolutely pure condition we are entirely unfamiliar with

1 Kaufman points out another important defect in the experiments indicating
a difference between the effects of poisons on enzymes and on cells, namely, that
in the experiments the concentration of enzymes is high, whereas in most cells
it is low. Solutions of trypsin stronger than 0.2 per cent, are not much
affected by toluol, thymol, etc., during twenty-four hours, but weaker solu-
tions are— those less than 0.02 per cent, being rendered inert. (Zeitschr.
f. physiol. Chem., 1903 (39), 434.)

2 It would not be profitable to discuss fully all the various theories and hypoth-
eses that have been advanced, but the reader is referred to the following
chief compilations of the entire subject : Oppenheimer, " Die Fermente una
ihre Wirkungen ," Leipzig, 1903, Effront, " Enzymes and their Applications,"'



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THE NATURE OF ENZYMES AND THEIR ACTIONS 63

their chemical characters, and consequently are obliged to
recognize them solely by their action. As far as we know, true
enzymes never occur except as the result of cell life — they are
produced within the cell, and increased in amount by each new
cell that is formed, and, furthermore, they are present in every
living cell without exception. As the same facts are equally
true of the proteids, and apparently true of nothing else, it is
natural to associate the enzymes with proteids, and so explain
the importance of the proteids for cell life. 1 If enzymes are
obtained in any of the usual ways from animal cells or secre-
tions they are always found to give the reactions for proteids,
even if repurified many times. But it is well known that
whenever proteids are precipitafed the other substances in the
solution tend to be dragged down by the colloids, and it is
possible that the enzymes are merely associated with the pro-
teids in this way. Furthermore, enzymes are known to become
so closely attached to stringy proteid masses, such as fibrin and
silk, that they cannot be removed by washing. Some have
claimed that they have secured active preparations of pepsin
and invertase that did not give proteid reactions and contained
very little or no ash or carbohydrate ; but it has so far been
impossible to secure trypsin free from proteid, and diastase
seems to be certainly of proteid nature. Analyses of enzymes
purified as completely as possible do not have great worth, for
the " purified " enzymes are probably far from pure ; however,
it is of some importance that they vary greatly in the propor-
tions of carbon, hydrogen, and nitrogen which they contain,
indicating that possibly different enzymes may be of very
different nature. Active gum enzymes, with oxidizing proper-
ties, are said to have been prepared free from nitrogen. 2 Macal-
lnni has shown microchemically that phosphorus is closely
associated with the formation of zymogen granules in cells,
which seem to be started in the nucleus ; and there are many
other observations suggesting that certain ferments are closely
related to the nucleo-proteids. This is particularly true of the
oxidases, which seem also to contain iron and manganese. A
final point of importance in support of the proteid nature of

translated by S. C. Prescott, New York, 1902. Reynolds Green, " Soluble
Ferments and Fermentation," 1901. In this chapter references will not
usually be cited unless they are from works published later than Oppenheimer's
book, in which all original work of importance can be found.

1 Another important point is that the closest imitation of enzymes, Bredig's
"inorganic ferments," seem to owe their action to their colloidal nature.

* A recent paper by Tschirsch and Stevens casts considerable doubt upon
this statement (Pharmac Centrhalle., 1905 (56), 501.)



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64 ENZYMES

enzymes is that pepsin destroys trypsin and diastase, while
trypsin destroys pepsin.

So uncertain, however, is our information concerning the
chemical nature of the enzymes, that it has become possible for
a hypothesis to be developed urging that enzymes are immate-
rial, that the actions we consider as characterizing enzymes are
the result of physical forces which may reside in many sub-
stances, and perhaps even free from visible matter. Arthus,
who has been the chief champion of this very interesting con-
ception, compares enzyme action to such forces as magnetism.
A magnetic iron bar loses its characteristic property when
sufficiently heated, just as an enzyme does. Dissolve the mag-
net or the enzyme in strong hydrochloric acid and they both
lose their power to affect other substances. It has been equally
impossible to isolate enzymes and magnetism, both of which
are recognized by their actions, and not by themselves. Just
as light, heat, and electricity were once considered as matter, so
has it also been with enzymes, and Arthus believes that they
will eventually be stricken from the list of material things and
considered as forms or a form of energy. There can be no
question that this conception rests on strong grounds, and it
possesses the stimulating qualities that make a hypothesis help-
ful, but, as Oppenheimer says, all chemical substances may be
considered in the same way. We recognize all bodies through
some form of energy ; if we speak of sulphuric acid, it is really
of the properties of energy it shows, such as its taste, which is
the energy imparted by its ions to the nervous system ; or its
combining with bases, etc., which also is a manifestation of
energy. In the same way we recognize the ferments, and we
may properly believe them to be fully as much definite sub-
stances as is sulphuric acid. The magnet comparison also
falls when we remember that the magnetism can be introduced
into a bar of iron and removed at will, but as yet it has not
been possible to introduce enzymatic properties into an inert
proteid, or to restore them to an enzyme that has been
destroyed by heat.

Another valuable piece of evidence of the material existence
of enzymes is their specific nature, lipase affecting only fats,
and trypsin only proteids, indicating chemical individuality.
They are true secretions, formed within the cell by recognizable
steps ; and, furthermore, when injected into the body of an ani-
mal, they give rise to the formation of specific immune bodies
that antagonize their action. Emil Fischer's work with the
sugar-splitting enzymes, moreover, indicates that they owe their



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THE PRINCIPLES OF ENZYME ACTION 65

action to their stereochemical configuration. He prepared two
sets of sugar derivatives which differed from each other solely
in the arrangement of their atoms in space (i. e. y isomers) and
found that one specific enzyme would split members of only
one of the varieties, while another enzyme would act only on
the variety with the opposite isomeric form. These experi-
ments make it very probable that there must be a certain
relation of geometrical structure between an enzyme and the
substances it acts upon, and leaves little question of its
material nature.

Bredig L has found that colloidal solutions of metals have
many of the properties of true enzymes, accomplishing many
of the decompositions produced by enzymes, being affected by
temperatures of nearly the same degree, and even being " poi-
soned" by substances that destroy or check enzymes. The
only possible explanation of these observations seems to be that
the enzyme effects are brought about by surface phenomena. A
colloidal solution of platinum, so far as is known, differs from
a piece of metallic platinum solely in the enormously great
amount of surface it offers in proportion to its weight, and it is
well known that surfaces may affect chemical action. Hence
we have the possibility that some enzyme actions, at least, may
depend upon the existence of a very large surface, and since by
no means all colloids are enzymes, that this surface must bear
a certain relation in form to the surface of the body that is to
be acted upon.

THE PRINCIPLES OF ENZYME ACTION

The effects produced by enzymes, which at one time were
considered quite unique and remarkable, have now been made
comparatively plain, chiefly through the observations of Ost-
wald on related chemical reactions ; and by the investigations
of Croft Hill, Kastle and Loevenbart, and others, on enzymes,
which show that enzyme action is in no way different from
chemical action observed independent of enzymes. The funda-
mental consideration is that chemical reactions are reversible,
that is, that their tendency is to establish an equilibrium, and
that the change may be from either side of the equation. The
action of enzymes is similar to that of all catalytic agents, that
is, they increase the speed of reaction. In the case of such a
reaction as that of NaOH and HC1, the reaction is so rapid
that the effect of catalyzers could hardly be noticed ; but with

l R65um£ in Ergebnisae der Physiol., 1902 (Bd. L, Abt. 1), p. 134; also
Bergell, Zeit. klin. Med., 1905 (57), 382.
5



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66 ENZYMES

many other substances the reaction is very slow, and without
the presence of catalyzers it would go on almost or quite
imperceptibly. For example, ethyl butyrate saponifies on the
addition of water according to the following equation :

C,H 6 - O - OC- C,H 7 + H f O +£ C^HjOH + HOOC - QH,.

On the other hand, if ethyl alcohol and butyric acid, the prod-
ucts of this reaction, are placed together, they will combine to
form ethyl butyrate ; in other words, the reaction is reversible,
as indicated by the arrows in the equation. In any event,
however, the reaction is not complete, but continues only until
a certain definite proportion of ethyl alcohol, butyric acid,
ethyl butyrate, and water exists, when the change will stop, t. e.,
equilibrium is established. The time that would be required for
this reaction to occur at room temperature would be extremely
long, the change being hardly noticeable, but in the presence
of a catalytic agent (which may be colloidal platinum or
lipase) the reaction goes on much more rapidly. Catalytic
agents, therefore, merely hasten reactions which would go on
without them, and they do not initiate or change the nature of
chemical reactions at all. When equilibrium is established, the
reaction stops and the enzyme has nothing more to do. Further-
more, and this is a recently appreciated fact, enzymes will has-
ten synthesis just as well as they hasten catalysis. Croft Hill
first showed that maltase would synthesize glucose into maltose ;
Kastle and Loevenhart soon after established the synthesis of
ethyl butyrate under the influence of lipase, and Neilson l
demonstrated that platinum black bad the same property.
Taylor 2 first synthesized one of the normal body fats, triolein,
by the action of lipase (from the castor-oil bean) upon oleic acid
and glycerin. It may seem improbable at first sight that the
synthesis of proteids can be accomplished by enzymes, as is the
relatively very simple synthesis of carbohydrates and fats, but
the improbability disappears when we recall the well-known
fact that the products of proteid splitting in passage through
the intestinal wall disappear and are reconverted either there or
elsewhere into body proteids. Proteids manifestly are synthe-
sized and we have not a little reason to believe that this is
accomplished by enzymes, presumably by a reversal of their
action in the establishment of equilibrium. Taylor was unable
to synthesize protamin, one of the simplest proteids, by the
action of trypsin upon its cleavage products, but it has been

1 Amer. Jour, of Physiol, 1903 (10), 191.

2 Univ. of California Publications (Pathology), 1904 (1), S3.



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THE PRINCIPLES OF ENZYME ACTION 67

found that the addition of proteolytic enzymes to solutions of
pure albumose leads to the formation of a jelly-like, insoluble
proteid substance, which seems to be the effect of a reversed
action on the part of the enzymes. 1 Indeed, the question has
been raised whether the coagulating or " lab-ferment " (rennin)
of the stomach is anything more than the pepsin itself, acting
in a reverse direction. 2 Another well-known synthetic action
that seems to be due to reversible ferment action is the forma-
tion of hippuric acid from benzoic acid and glycocoll in the
kidney ; the formation of glucose into glycogen and its reforma-
tion are also probably both accomplished by one and the same
enzyme acting reversibly. Other reversible reactions less
closely related to animal cells have also been described.

The reversible nature of enzyme action explains many prob-
lems of metabolism, and makes the whole field much clearer.
The following consideration of the newer understanding of fat
metabolism on this basis may explain the manner in which
chemical changes are believed to occur in the cells and fluids
of the body : 3

In the intestines fat is split by lipase into a mixture of fat, fatty acid, and
glycerin ; but as the fatty acid and glycerin are diffusible, while the fat is
not, they are separated from the fat by absorption into the wall of the intes-
tine. Hence an equilibrium is not reached in the intestine, so the splitting
continues until practically all the fat has been decomposed and the products
absorbed. When this mixture of fatty acid and glycerin first enters the epi-
thelial cells lining the intestines there is no equilibrium, for there is no fat
absorbed with them as such. Therefore the lipase, which Kastle and Loeven-
hart showed was present in these cells, sets about to establish equilibrium by
combining them. As a result we have in the cell a mixture of fat, fatty acid,
and glvcerin, which will attain equilibrium only when new additions of the two
last substances cease to enter the cell. Now another factor also appears, for
on the other side of the cell is the tissue fluid, containing relatively little fatty
acid and glycerin. Into this the diffusible contents of the cell will tend to pass
to establish an osmotic equilibrium, which is quite independent of the chem-
ical equilibrium. This abstraction of part of the cell contents tends to again
overthrow chemical equilibrium, there now being an excess of fat in the cell.
Of course, the lipase will, under this condition, reverse its action and split the
fat it has just built into fatty acid and glycerin. It is evident that these proc-
esses are all going on together, and that, as the composition of the contents of
the intestines and of the blood-vessels varies, the direction of the enzyme action
will also vary. In the blood-serum, and also in the lymphatic fluid, there is
more lipase, which will unite part of the fatty acid and glycerin, and by re-
moving them from the fluid about the cells favor osmotic diffusion from the
intestinal epithelium, thus facilitating absorption.

Quite similar must be the process that takes place in the tissue cells through-
out the body. In the blood-serum bathing the cells is a mixture of fat and its

1 Heraog, Zeit. f. physiol. Chem., 1903 (39), 305.

1 The results of nitration experiments suggest that pepsin and rennin are
distinct substances (Levy, Jour. Infect. Diseases, 1905 (2), 1 ; also see Schmidt-
Nielsen, Zeit physiol. fchem., 1906 (48), 92).

3 See Loevenhart, Amer. Jour, of Physiol., 1902 (6), 331 ; Wells, Journal
Amer. Med. Assoc, 1902 (38), 220.



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68 EXZYMES

constituents, probably nearly in equilibrium, since lipase accompanies them.
If the diffusible substances enter a cell containing lipase, e. g. t a liver cell, the
process of building and splitting will be quite the same as in the intestinal epi-
thelium. The onlpr difference is that here the fatty acid may be remoTed
from the cell by being utilized by oxidation or some other chemical transforma-
tion.

To summarize, it may be stated that throughout the body
there is constantly taking place both splitting and building of
fat. Fat enters the cells, leaves them, and is utilized only in
the form of its acid and alcohol, never as the fat itself. Fat
constitutes a resting stage in its own metabolism.

If proteolytic enzymes are also reversible, then the phenom-
ena of proteid metabolism are similarly explained, for there
is no doubt that every cell and body fluid contains proteolytic
enzymes.

AH metabolism, then, may be considered as a continuous at-
tempt at establishment of equilibrium by enzymes, perpetuated by
prevention of attainment of actual equilibrium through destruction
of some of the participating substances by oxidation or other chem-
ical processes, or by removed from the cell or entrance into it of
materials which overbalance one side of the equation.

In just what manner the enzymes accomplish their catalytic
effect is yet unknown. A favorite idea is that they form loose
compounds with the body to be split and with water ; the result-
ing compound being unstable and breaking down, the water
remaining attached to the components of the substance. There
is some evidence, but not conclusive, indicating that the enzyme
does enter into combination with its object. Euler has suggested
that enzymes increase ionization, which is at the bottom of the
chemical changes.

Enzymes do not act catalytically on all substances by any
means, but show a decidedly specific nature. They affect only
organic substances, and the actions are limited to two processes —
hydrolysis and oxidation, or the reverse processes of dehydration
and reduction. ! The most essential difference between the
enzymes and the chemicals that can accomplish hydrolysis or
oxidation is this : the ordinary chemical reagents produce their
effects on many sorts of substance, the enzymes are specific ;
thus hydrochloric acid will hydrolyze starch or proteid with
equal facility, but pepsin will «ot affect starch at all.

The very specific nature of the enzymes, their activation by
other body products, the fact that they seem to be bound to the
substance upon which they act, that they are susceptible to heat,

1 Alcoholic fermentation may be an exception, the change being CjH u 6 "—
2C a H 5 OH -j- 2CO„ but it is very possibly an intramolecular oxidation.



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THE PRINCIPLES OF ENZYME ACTION 69

and that they produce immune bodies when injected into exper-
imental animals, all suggests the probability of a relationship
between enzymes and toxins. This matter will be discussed more
fully in considering the chemistry of immunity against enzymes.

General Properties of Enzymes. — Other properties of
enzymes may be briefly mentioned. The speed of reaction they
produce increases with the amount of enzyme present, but not
in direct proportion (except with rennin). Very dilute acids
favor the action of nearly all ferments, and alkalies are unfa-
vorable for all but trypsin, ptyalin, and a few others. Weak
salt solutions also are more favorable than distilled water.
(These facts suggest strongly the possibility that ions play an
important r6le in the process.) Water and dilute glycerin dis-
solve enzymes, which form always colloidal solutions that are
very slightly dialyzable ; and they may be precipitated from
solution by alcohol, and redissolved again with but slight im-
pairment of strength. Filtration through porcelain filters is
not complete, from 10 to 25 per cent, of most enzymes being
lost in each filtration. l As before mentioned, many chemicals
poisonous to bacteria have little influence on most enzymes, but
nearly all substances when concentrated are injurious or destruc-
tive, and some enzymes are known that are more susceptible to
antiseptics than are the cells that contain them. Formaldehyde
is very destructive to enzymes, even when dilute. The effect of
proteid-coagulating antiseptics upon enzymes is, of course,
greatly modified by the amount of proteid substances mingled
with the enzymes ; and the effects of heat and other injurious
influences are greatly decreased by the presence of proteids and



Online LibraryH[arry] Gideon WellsChemical pathology; → online text (page 6 of 57)