An Interview with Manuel Varela and Ann Varela: About Maud Menten—One of Canada’s Finest!

Jun 4, 2020 by

Maud L. Menten

Michael F. Shaughnessy –

1) Maud L. Menten was one of the first women physicians in Canada—a laudable achievement—but what about her roots? What were her parents like?

Dr. Maud (Maude) Leonora Menten is best known for her pioneering biochemistry work in the field of enzymology. She was born March 20, 1879, in Port Lambton, Ontario, Canada. Menten grew up in Harrison Mills, British Columbia, Canada. Menten’s father William was a boat pilot on the Fraser River neighboring Chilliwack, near Vancouver, British Columbia. Her mother was employed as a postmistress. Menten’s parents also were thought to have owned and operated a general store and a hotel.

2) She was fortunate enough to attend some of the excellent schools of her time. Can you tell us where she went to school and what her accomplishments were at the time?

In 1900, Menten traveled to Toronto, Canada, to attend the University of Toronto, where she took her B.A. degree in 1904. She then entered graduate school at the same institution. Her MS thesis project involved studying the distribution in the brain of chloride-based compounds. At the University of Toronto, Menten took her master’s degree in physiology in 1907.

That same year, Menten accepted a fellowship at the Rockefeller Institute for Medical Research. Menten worked as an intern for one year at the New York Infirmary for Women and Children. Menten returned to the University of Toronto in Canada and qualified as a medical doctor, earning her M.D. degree in 1911.

In 1912, Menten worked with the prominent surgeon George Crile on the control of acid-base balance during anesthesia. The year that the Titanic sunk in 1912 was the same year that Menten traversed the ocean on route to Berlin, Germany, to work with Leonor Michaelis and co-authored their classic paper in Biochemische Zeitschrift. In 1916, Dr. Menten received her Ph.D. at the University of Chicago. Her dissertation title was “The Alkalinity of the Blood in Malignancy and Other Pathological Conditions; Together with Observations on the Relation of the Alkalinity of the Blood to Barometric Pressure.”

3) She spent some time in New York, working at the Rockefeller Center, where she developed some of the work of Marie and Pierre Curie in terms of the treatment of tumors. 

In 1907, due to her undergraduate and graduate academic record, Menten became a pathology scholar at the Rockefeller Institute of Medical Research, New York City, New York, in the United States. Menten studied in laboratory rats the effects on the growth of transplantable carcinogenic tumors from radium bromide exposure. At the time, radium and its derivatives were entirely new to the scientific world, as Marie Skłodowska and Pierre Curie had discovered the element only a few years prior.

Working with two other scientists, Simon Flexner and James Jobling, she published the results of the experiments in 1910, producing the Rockefeller Institute’s first known monograph titled “Experiments on the influence of radium bromide on a carcinomatous tumor of the rat.” The work would eventually become important for the development of so-called radiobromide as a cancer treatment possibility.

4) The Michaelis-Menten formula—why is this important? (Moreover, apparently, she crossed the Atlantic during the year the Titanic sunk, to develop this concept!)

Moving to the laboratory of Dr. Leonor Michaelis at Berlin Municipal Hospital in Germany, in 1912, Dr. Menten made one of the most significant scientific discoveries ever in the field of biochemistry. There is no doubt that the basic principles of enzyme biochemistry established by the pioneering work of Drs. Menten and Michaelis will last for millennia.

Menten and Michaelis published their famous 1913 paper in Biochemische Zeitschrift, which translates from German to Biochemical Journal. The title of the now-classic article is Kinetics of Invertase Action. During the extensive course of their experimental studies, they measured the activity of an enzyme, which at the time, they had called a ferment. The protein is known as invertase, and it catalyzes the conversion of table sugar sucrose into fructose and glucose. The name invertase refers to the fact that the enzyme’s action results in the inversion of the optical rotation property from a positive orientation of the sucrose substrate into a negative rotation representing the products of the enzyme protein.

Menten herself measured the invertase activity by monitoring the optical rotation with time as the reaction proceeded from sucrose to the production of fructose and glucose. She tested the hypothesis that the enzyme formed a complex of invertase bound to sucrose, a disaccharide, degrading sucrose into two monosaccharides. She further hypothesized that as the invertase activity proceeded, the rate of optical rotation inversion rate was proportional to the concentration of enzyme-substrate complexes formed.

Menten and Michaelis understood the products of the enzyme, fructose, and glucose monomers were inhibitory to the protein, a phenomenon now known as feedback inhibition. Thus, they measured the initial rates of the enzymatic reaction to avoid confounding inhibitory effects. These initial rate measurements, i.e., the enzyme’s reaction velocities, proved to be beneficial to their success in establishing their pioneering discoveries.

They further postulated that at the start of the reaction, the enzyme (E) combines with the substrate (S) to form the E-S complex reversibly. Then, when the biochemical reaction is complete, E-S complex falls apart to free up the enzyme E and the product P. The enzyme is now free to undergo another reaction of the same type but with another molecule of the substrate.

Using a kinetics approach, they developed an ingenious means for characterizing the parameters of enzyme activities. They proposed a new term called the Michaelis constant, Km, to help explain the various rates and numbers of each of the steps in the formation the E-S, or free E, S, and P from their various associations during enzyme catalysis. Considering initial velocity, V0, and the concentration of the substrate, one could use the Michaelis-Menten equation to determine the Vmaxfor a given enzyme’s action. From these biochemical associations, they were also able to derive the so-called Michaelis constant, called Km, mathematically.

Michaelis-Menten Equation

Later, Menten and others were also able to experimentally measure the enzyme activities and characterize the Vmax and Km values using the Double-Reciprocal or Lineweaver-Burke plot (see the chapter on Michaelis).

Reciprocal MM equation

https://upload.wikimedia.org/wikipedia/commons/9/9b/Reciprocal_MM_equation.JPG

https://upload.wikimedia.org/wikipedia/commons/9/9b/Reciprocal_MM_equation.JPG

Figure Reciprocal Michaelis-Menten equation

The graphical method for evaluating kinetic data from enzyme activity testing was developed by Hans Lineweaver and Dean Burk in 1935. The Lineweaver-Burke approach became useful for assessing the interaction between enzymes and their inhibitors. It became possible to determine whether inhibitors of enzymes worked in competitive versus non-competitive manners.

https://upload.wikimedia.org/wikipedia/commons/thumb/e/ed/Lineweaver-Burke_plot_competitive_inhibition.svg/1280px-Lineweaver-Burke_plot_competitive_inhibition.svg.png

https://upload.wikimedia.org/wikipedia/commons/thumb/e/ed/Lineweaver-Burke_plot_competitive_inhibition.svg/1280px-Lineweaver-Burke_plot_competitive_inhibition.svg.png

Figure Lineweaver-Burke plot competitive inhibition

Considering the work of Menten and Michaelis and their kinetic and catalytic parameters for enzymes, the results could be exploited to evaluate whether an enzyme inhibitor is competitively binding to the active site. Typically, the active site of an enzymatic catalyst is the location within the protein where the actual biochemistry is taking place. Therefore, if an inhibitor of the enzyme also binds to the same area where the substrate binds and somehow interrupts the biochemical reaction at this active site, then the enzyme’s catalytic activity is inhibited directly. Thus, determining whether an inhibitor is competitive in its regulatory nature, i.e., it competes with the substrate for the critical active enzyme site, can be quite valuable.

First, the mechanical nature of the biochemistry that occurs within the active site may be understood at the molecular level. One may then construct mutations at or near the active site to aid in the identification of the critical location of enzyme action. Second, the enzyme’s inhibitor may be tweaked with sight structural modifications to enhance the inhibitor’s activity upon the catalyst.

https://upload.wikimedia.org/wikipedia/commons/thumb/a/aa/Lineweaver-Burke_plot_non-competitive_inhibition.svg/514px-Lineweaver-Burke_plot_non-competitive_inhibition.svg.png

https://upload.wikimedia.org/wikipedia/commons/thumb/a/aa/Lineweaver-Burke_plot_non-competitive_inhibition.svg/514px-Lineweaver-Burke_plot_non-competitive_inhibition.svg.png

Figure Lineweaver-Burke plot non-competitive inhibition

Conversely, if an enzyme’s inhibitor is non-competitive, then it suggests that it binds elsewhere. In the case of a non-competitive inhibitor, wherever the location is on an enzyme to which it is binding, it will likely NOT be at the enzyme’s active site. The inhibitor can perhaps bind an allosteric site to regulate the enzyme action. An enzyme’s active biochemistry site is essential to know because the mechanistic nature of an enzyme’s given activity is further understood to the molecular level.

Therefore, to determine whether an inhibitor is competitive, one merely needs to measure the enzyme activity experimentally in the presence of various concentrations of substrate and inhibitor. If the experimenter observes a significant change in the Km value but not the Vmax value, then it is known that the inhibitor is competitive in its temperament. On the other hand, if the Km remains the same but the Vmax is changed, then the inhibitor is non-competitive.

Such knowledge may be further used to construct mutations on these non-competing sites for tweaking enzyme activities. It can permit biochemists to focus on altering the active sites so that new substrates are considered by an enzyme. These new substrates could be starting substrates that are perhaps toxic and need to be degraded, effectively reducing their potential toxic effects.

Alternatively, one might be permitted to alter the active site of an enzyme so that entirely different substrates are used to enhance the production of needed products for biotechnological, industrial, or medical use. Since enzymes are known to be the chief players in all known metabolic reactions conducted by living beings, the potential here is virtually limitless.

5) Enzyme kinetics—what are they, and why are they important?

Menten’s medical research with Leonor Michaelis in 1913 yielded the famous Michaelis-Menten equation, one of the essential concepts in enzyme kinetics. The term enzyme kinetics refers to the velocities by which biochemical reaction takes place in living cells that make up whole organisms. In all living beings, the rates of the chemical reactions catalyzed by protein enzymes constitute their dynamic behaviors. These reaction rates or velocities are further characterized by measuring the amounts of products made by catalytic enzymes as a function of time. Analysis of such kinetic (rate) data is essential for assessing the structures, catalytic mechanisms, functions, and regulation of enzymes. Such enzymatic knowledge, in turn, can be quite significant for applications regarding medical, industrial, and biotechnological purposes.

The classic publication of the 1913 paper by Menten and Michaelis may be considered the starting point for the eventual establishment of enzyme kinetics as a bona fide field of biochemistry. Indeed, in modern times, enzymes and their kinetic functions are given prominent attention in an excellent textbook on general biochemistry. The relevance of Menten’s studies of enzyme kinetic behavior is that such proteins often adhere nicely to the Michaelis-Menten equation and its derived Michaelis constant, Km.

Menten’s kinetic studies considered an assumption that as the biochemical reactions proceed, they reach an equilibrium. That is, during a biochemical enzyme-catalyzed reaction, the concentrations of the enzyme and the substrate reach an equilibrium with the E-S complex concentration. To invoke the assumption, Menten and Michaelis had built on the earlier work of Dr. Victor Henri, thus, postulating that an enzyme binds to and forms a complex with the substrate, E-S. While not entirely accurate, the equilibrium assumption led to the discovery of the notion of the E-S, and it was an important finding. In modern times, thus, E-S is also referred to as the Michaelis complex.

Another consideration that was invoked to build on the kinetic work of Menten and Michaelis was that of the so-called steady-state assumption. This consideration was briefly mentioned in the chapter dealing with Michaelis, and the assumption states that as the formation of the E-S complex occurs, it stays relatively unchanged as it corresponds to the disappearance of the substrate and appearance of the enzyme’s product at the end of the reaction. The steady-state assumption, therefore, predicted that the E-S concentration is a constant entity. It is necessary to emphasize that a steady-state situation does not necessarily mean that the reaction components are at equilibrium.

The lack of such an equilibrium state is because the respective concentrations of substrate and product are rapidly changing entities, even though the concentration of free enzyme and Michaelis complexes (E-S) are substantially consistent in their respective amounts.

Nevertheless, despite these qualifications, the above two assumptions employed by Menten and Michaelis proved to be extremely useful toward the development of modern reaction mechanism kinetics and enzyme chemistry in general. Such Menten-Michaelis kinetics is of tremendous relevance in the transport of solutes such as metabolites or of antimicrobial agents across the membrane. All organisms acquire nutrients and expel inhibitory agents via transport across the membrane. These transport systems very often follow Menten-Michaelis kinetics, and as such, can be regulated via inhibitors, as described above.

6) Dyes—we take them for granted today—but what was her role in discovering them? Or using them to investigate the human body?

Menten had employed the use of so-called azo-dye coupling reactions to investigate the role of an important enzyme known as an alkaline phosphatase in the kidney. This groundbreaking work was published in 1944. Since its discovery, Menten’s coupling reaction system has been used as a routine tool for diagnosis of medical disease and in fundamental biochemical research studies.

The work led to the establishment of the new field of biochemistry called enzyme histochemistry. One avenue that opened as a direct result of Menten’s discovery involved glycogen histochemistry. She also investigated nucleic acid histochemistry in the bone marrow.

Menten’s work was referred to as a “stroke of genius” by Professor Anthony Guy Everson Pearse in his first edition of the 1953 book titled Histochemistry. Pearse had given credit to Menten for initiating the new field of enzyme histochemistry.

The new work facilitated the ease with which investigators could detect desired proteins or other molecules within localized areas of the cell or tissues by fusing alkaline phosphatase to them and examining their loci with chromogenic dyes. Structural studies of membrane transport proteins could be studied at the molecular level with alkaline phosphatase fusions with elements of the transporters. In each of these cases and others, Menten’s enzyme histochemistry approach is still used in modern times.

7) Blood sugar—we hear so much about it today—but apparently, she researched it early on. What did she find?

In 1924, Helen M. Manning and Maud Menten at the University of Pittsburgh discovered that bacterial infection produced by various serovars of Salmonella enterica enhanced the levels of the sugar glucose in the blood, a condition called hyperglycemia. In Menten’s day, the illnesses caused by Salmonella enterica strains were called enteritidis-paratyphoid B. The diseases caused by bacterial variants were called typhoid fever, gastroenteritis, food poisoning, and septicemia. Today we refer to each of these illnesses as a form of salmonellosis.

Menten and Manning wrote in their historical paper that the causative agents of enteritidis and paratyphoid B were Bacillus enteritidis and Bacillus paratyphoid, respectively. Later, these microbes were changed to Salmonella enteritidis and Salmonella paratyphoid. More recently, as genomes projects revealed that these “species” were all primarily the same one, microbiologists then referred to all of them collectively as Salmonella enterica. Consequently, the specific epithets enteritidis and paratyphoid were later relegated to serovars (also called serotypes).

Manning and Menten injected rabbits, dogs, rats, and guinea pigs with infectious doses of Salmonella enterica serovars Enteritidis or Paratyphoid, among other bacteria, and they permitted the illnesses to manifest themselves. The animals were food-deprived before blood samples were taken for measurement of antiserum, nitrogen, and glucose levels. After the laboratory animals died or were killed, necropsies (autopsies for non-human animals) were performed, and their organs and tissues were tested for the types of bacteria possibly growing in them. The infecting bacteria were then identified to the species level. Lastly, the levels of blood glucose were compared with the infective numbers and types of bacteria found.

Manning and Menten found histological lesions produced by the infecting Salmonella enterica. Interestingly, they found that the livers and kidneys from the dead animals were primarily affected by the infection. Furthermore, the animals from which their tissues had been detrimentally affected by the Salmonella also showed initially high glucose in the blood, followed by an abnormally low blood glucose amount. In some cases, the kidneys were damaged by infection, resulting in a possible disturbance in the ability to excrete nitrogen-containing waste products. Menten and Manning reported that the liver damage was the main pathological finding following bacterial infection. Importantly, they discovered that Salmonella infection caused hypoglycemia.

The groundbreaking work proved to be important because Menten and Manning discovered that infection with the Salmonella enterica serovars Enteritidis or Paratyphoid disturbed the liver storage capacities of glycogen, effectively depleting the storage reserves of glycogen. Thus, they had established that Salmonella pathology meant an abnormal glycogen reduction in the liver.

Later studies performed in the early 1970s wholly confirmed the 1924 work of Dr. Menten and her colleague Helen Manning. The newer studies had identified an effect by the Salmonella bacterial toxins upon certain liver glycogen synthase enzymes.

8) She is credited with doing the first separation of blood hemoglobin proteins by electrophoresis—why is this important?

Electrophoresis is a standard biochemistry-based technique used in laboratories worldwide to separate biomolecules. The method is used not only for protein separation, but also for that of nucleic acids, sugars, or even fats. Further, electrophoresis can be used to separate and detect combinations of biomolecules, such as nucleoproteins, glycoproteins, glycolipids, lipoproteins, RNA-DNA hybrids, etc.

In 1944, Menten, working with Marie A. Andersch and Donald A. Wilson at the University of Pittsburgh, published a paper dealing with the sedimentation constants and mobilities of hemoglobin variants called carbonylhemoglobin from human fetal and adult blood. When oxygenated hemoglobin loses its oxygen, it is referred to as the deoxygenated form of the blood protein. These deoxygenated hemoglobin molecules may be susceptible to binding by carbon dioxide or carbon monoxide.

The carbonylhemoglobin species is a hemoglobin molecule that is devoid of oxygen but bound to carbon monoxide in its place. Menten and colleagues had hypothesized that distinctive species of carbonylhemoglobin could be differentiated between fetal and adults.

Menten and co-workers took blood samples from adult humans, infants who were five-, nine-, and 90-days old, and from umbilical cords. To the blood samples, they added citrate buffer saturated with carbon monoxide, washed the blood by centrifugation, placed in a mixture of phosphate buffers, and dialyzed to place the spent buffers with new solutions saturated with carbon monoxide. Next, they measured the electrical conductivity and took pictures to record the movement, i.e., electrophoretic mobility, of the carbonylhemoglobin molecules.

Then, they determined the sedimentation constants by using high-speed ultra-centrifuges and taking new pictures of the carbonylhemoglobin mobilities along the way. Lastly, a series of calculations were performed to acquire the blood protein mobility data and their respective sedimentation constants. The entire process was arduous, labor-intensive, and time-consuming to work up each of the various blood samples.

From the various electrophoretic and sedimentation analyses, Menten and colleagues were able to discern that human fetal and adult hemoglobin molecules consisted of two distinctive variants. Thus, not only were they the first to conduct an electrophoretic analysis of human hemoglobin, but they also managed to sort out the hemoglobin derivatives and distinguish between fetal and adult blood hemoglobin. In being the first to characterize hemoglobin electrophoretically in 1944, the Menten team beat out the famous Linus Pauling by five years.

Although Menten and her associates were the first to use the widely used method to sort out the biochemical behaviors of human hemoglobin, Pauling mistakenly got the bulk of the credit for it. Historians of science attribute this discrepancy in credit to Pauling’s outspokenness and celebrity status amongst the scientific community.

9) On YouTube is a rather comical demonstration of the Menten ideas—and they refer to it as Elephant’s toothpaste? How does this 5-minute demonstration elucidate and clarify Dr. Menten’s work?

There are indeed many a YouTube video demonstrating the Elephant’s toothpaste phenomenon, generating a sizeable foam-like substance from a few pure chemicals. Scientists who narrate these remarkable videos explain the catalytic nature of the reactive process. The demonstrations consist of adding a few items and watching the rapid formation of a giant glob and taking the form of a toothpaste-like material suitably big enough for that of an elephant. Of course, the material formed is not necessarily suitable for teeth brushing by humans or elephants; often, the material formed is quite bubbly and harbors oxygen and water. The general reaction entails adding a few ingredients like soap (to generate the foam action), hydrogen peroxide, and a catalyst, which can consist of a chemical, like potassium iodide, or a microbe, like yeast.

The yeast can harbor the enzyme catalase, which rapidly converts the hydrogen peroxide into oxygen and water. The catalase enzyme follows the classic Menten-Michaelis principles of enzyme action. Further, the enzyme happens to be one of the fastest enzymes known to science; hence, the name catalase. Humans carry catalase in the blood, and when antiseptics that contain hydrogen peroxide is added to a skin injury, the patient will frequently see a quick bubbling process emerging from the affected area. This foaming action is the result of the production of oxygen and water from the hydrogen peroxide substrate due to the catalytic action of catalase.

10) What have I neglected to inquire about this pioneering female scientist, painter, and researcher from Canada?

Although Dr. Maud Menten was among the first women in Canada to earn a medical doctorate, she did not acquire promotion to full professor until 1948 at the age of 70. Before that year ended, Menten retired.

From 1923 to 1950, Menten’s career in pathology at the University of Pittsburgh started as an assistant professor of pathology and then Associate Professor in the School of Medicine, and later promoted to chair of the pathology department at the Children’s Hospital of Pittsburgh. At Pittsburgh, Menten had numerous opportunities to collaborate with other prominent colleagues. Menten was extremely busy with these two positions but somehow balanced her time to author and co-author more than 70 publications. Her final academic position was as a research fellow at the British Columbia Medical Research Institute from 1951-1953. Her studies focused on cancer research.

Amongst her other significant accomplishments was the development of a scarlet fever vaccination program using Streptococcus bacteria during the 1930s and early 1940s.

She spoke at least six languages. Menten enjoyed many adventurous and artistic hobbies. For example, she was musical and played the clarinet, a talented artist, perused outdoor challenges such as mountain climbing, adventurous with participating in an arctic expedition, and appreciated astronomy. Menten drove a Model-T Ford through the University of Pittsburgh area for nearly 32 years. It has been rumored over the years that the town residents recognized her car and got out of her way, as her driving skills remained at the novice level.

In 1998 ,Menten was posthumously inducted into the Canadian Medical Hall of Fame. The University of Toronto honored Menten with a permanent plaque. At the University of Pittsburgh, a portrait of Menten is displayed. Further, memorial lectures in her honor and an endowed chair were established in her name at Pittsburg. On July 17, 1960, Dr. Menten passed away in Leamington, Ontario, Canada.

For further information regarding this genuinely remarkable scientist, visit the following links:

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