Presidents Elected Without Winning the Popular Vote

Presidents Elected Without Winning the Popular Vote Five U.S. presidents have taken office without winning the popular vote. In other words, they did not receive a plurality regarding the popular vote. They were elected, instead, by the Electoral College- or in the case of John Quincy Adams, by the House of Representatives after a tie in the electoral votes. They were: Donald J. Trump, who lost by 2.9 million votes to Hillary Clinton in the  2016 election.George W. Bush,  who lost by 543,816 votes to Al Gore in the  2000 election.Benjamin Harrison,  who lost by 95,713 votes to  Grover Cleveland  in 1888.Rutherford B. Hayes, who lost by 264,292 votes to Samuel J. Tilden in 1876.John Quincy Adams,  who lost by 44,804 votes to  Andrew Jackson  in 1824. Popular vs. Electoral Votes Presidential elections in the United States are not popular vote contests. The writers of the Constitution configured the process so that only the members of the House of Representatives would be elected by popular vote. The Senators were to be selected by state legislatures, and the president would be selected by the Electoral College. The Seventeenth Amendment to the Constitution was ratified in 1913, making the election of senators to occur through popular vote. However, presidential elections still operate under the electoral system. The Electoral College is made up of representatives who are generally selected by the political parties at their state conventions. Most states except Nebraska and Maine follow a winner-take-all principle of electoral votes, meaning that whichever partys candidate wins a states popular vote for the presidency will win all of that states electoral votes. The minimum electoral votes a state can have is three, the sum of a states senators plus representatives: California has the most, with 55. The Twenty-Third Amendment gave the District of Columbia three electoral votes; it has neither senators nor representatives in Congress. Since states vary in population and many popular votes for different candidates can be quite close within an individual state, it makes sense that a candidate might win the popular vote across the entire United States but not win in the Electoral College. As a specific example, lets say the Electoral College is only made up of two states: Texas and Florida. Texas with its 38 votes goes entirely to a Republican candidate but the popular vote was very close, and the Democratic candidate was behind by a very small margin of only 10,000 votes. In the same year, Florida with its 29 votes goes entirely to the Democratic candidate, yet the margin for the Democratic win was much larger with the popular vote win by over 1 million votes This could result in a Republican win at the Electoral College even though when the votes between the two states are counted together, the Democrats won the popular vote. Calls for Reform In general, it is very rare for a president to win the popular vote yet lose the election. Although this has only happened five times in U.S. History, it has occurred twice in the current century. In 2016, Donald Trump lost the popular election by nearly 3 million votes, about 2% of the total votes cast. Discussion for reform of the Electoral College dates to the first years of the Constitution and has frequently been the topic of scholarly discussion. Defenders of the Electoral College argue that its violations of majority rule are an example of constitutional provisions that require super-majorities to take action. The Electoral College allows a minority to take an action- that is to select a president- and it is the only device of its kind in the Constitution. The primary way to change it is to amend the Constitution. Since the way states count votes can affect who wins and who loses, electoral reform is by its nature political: if a party is in power, the method used to get it there is not likely to be a target of change. Despite the current climate in that Democrats support change while Republicans do not, scholars believe that the situation is only temporarily one that swings towards one party or another: One proposal called the National Popular Vote Interstate Compact (NPVIC) is a sub-national reform to the Electoral College in which states agree to commit their electoral votes, as a unit, to the winner of the aggregate, national vote.  Sixteen states have signed on to date, some of which are Republican-controlled. One major purpose of the Electoral College was to balance the power of the electorate so that votes in states with small populations would not (always) be overpowered by larger-populated states. Bipartisan action is required to make its reformation possible. Sources and Further Reading Bugh, Gary, ed. Electoral College Reform: Challenges and Possibilities. London: Routledge, 2010.Burin, Eric, ed. Picking the President: Understanding the Electoral College. University of North Dakota Digital Press, 2018.Colomer, Josep M. The Strategy and History of Electoral System Choice. The Handbook of Electoral System Choice. Ed. Colomer, Josep M. London: Palgrave Macmillan UK, 2004. 3-78.Goldstein, Joshua H., and David A. Walker. The 2016 Presidential Election Popular-Electoral Vote Difference. Journal of Applied Business and Economics 19.9 (2017).Shaw, Daron R. The Methods Behind the Madness: Presidential Electoral College Strategies, 1988–1996. The Journal of Politics 61.4 (1999): 893-913.Virgin, Sheahan G. Competing Loyalties in Electoral Reform: An Analysis of the U.S. Electoral College. Electoral Studies 49 (2017): 38–48.

Western Art History Essay Example | Topics and Well Written Essays - 1250 words

Western Art History - Essay Example On the end view there is reflection on the mirror on the wall behind picture of the king and queen, the paradox is clear as it can be interpreted that the artist is trying to portray the royal couple, however in in-depth view it is seen that the kings daughter was the main focus. This painting by Diego Velazquez is one of the best painting works analyzed in the western paintings because of its complexity and confusing aspects that complicates its ease of understanding. Some of the enigmatic aspects of the painting that raise more questions about the painting include; the undetermined connection between the subjects in the picture and the viewers and the question on the picture being real or just mere illusions (Kleiner 317-326). The relationship that exist between the first view of the picture and the subjects on the picture is clear and can be easily analyzed, the main subject in the painting the king’s eldest daughter Infanta Margaret Theresa is at the center of the portrait and on her side are her maids surrounding her giving out clearly the picture of her being the maid of honor (Kleiner 330-337). Behind the daughters picture on the reflective mirror is the picture of the king and the queen who confusingly appear to be left out of the picture but again seem main subjects of focus. There is provision for vision beyond the front studio with the door that is on the back wall of the painting that confuses whether the character is headed to the studio front or headed away from the studio. I agree and support the argument that the painting is a classical master piece, Diego’s painting gives confusing interpretation that makes understanding of the idea communicated in the picture complex. The artist uses techniques in painting like the use of light and the picture on the reflective mirror to bring out the technique of picture within picture these inclusions of art work makes the picture one of the

Student Essay Topic

Student Essay TopicThe Hamlet Act V Essay Topics is to give students an opportunity to delve into the actual events of Hamlet. This is a great place for the students to learn how different characters fit into the rest of the play.Of course the inclusion of a character study in the Hamlet Act is only a small part of the selection process, there are several other aspects as well that must be considered. When you choose to use the Hamlet Act as a resource for essay topics, make sure you give attention to your choice of essay topic. You don't want to be wowed by the beauty of your chosen topic only to find out later that it was the wrong one.One thing to consider is whether you want to include the Hamlet episode in the text. Many people want to make the appropriate political statement with the use of the Hamlet Act. But do you want to appear uneducated, or do you want to have that wonderful Shakespearean quote that helps put the play in perspective?Some essay topics can use a hint of dra ma, while others may be quite serious. Consider the personality of the author. For example, if the author is a woman, you might want to add some slight drama to the topic.For some students the essay topics may not include the entirety of the play. If you do this, it is important to make sure you do something that uses the entire text. Also, a little extra attention can go a long way in making the reader understand the theme of the play.When the essay topics have been narrowed down, it is important to know what type of essay they will be. There are two types of essays that are used, thesis and summary. If you are unsure which type you should choose, the first thing to do is find a teacher or a mentor.Of course the main point is to use the essays to help you pass your ACT or SAT exams. While the essay topics are the least important part of the application, it is a good idea to have an idea of what is coming before you start the process.

The Battle Over College Essays - Part I -Common App Core Essay - Writing Eye Catching Essay and How to Win It

The Battle Over College Essays - Part I -Common App Core Essay - Writing Eye Catching Essay and How to Win It The True Meaning of College Essays - Part I - Common App Core Essay - Writing Eye Catching Essay Plagiarism checker software offers you proof that you haven't plagiarized. The premade tool is an appropriate layout made available to help you become through the planning stage of the e-Learning system, which makes it feasible to join elements with each other to create an ideal system. That's the reason why there are ready-to-use digital storyboard templates which may be gettable. Aside from plagiarism, it's important to follow academic tradition and adhere to all demands. Take into consideration the principal ideas that you need to bind with each other to make a single huge idea. They could utilize a paid tool like Copyscape or even use a complimentary plagiarism checker to detect if there's any present. To finish an assignment the most suitable way you need to stick to the format. A History of College Essays - Part I - Common App Core Essay - Writing Eye Catching Essay Refuted Wasielewski calls for extra research to totally explore a university's options but says using AI to improve the human-centered work of creating admission decisions could be something we start to see later on. I'm choosing these examples dependent on the points mentioned previously. To assist you craft the very best cover letter, we're supplying you with a few examples to reassess. It's vital that you customize each cover letter to the job that you're applying. Ask other people to score it by utilizing the Headline Scorecard. They know their preferred enrollment, then must figure out the number of acceptance letters they must put, knowing a lot of their choice applicants have other offer letters to think about. After all, each position values different experiences and responsibilities, and you are going to want to showcase different circumstances and career highlights based on the particular job you're seeking. Conducting research to finish an academic writing assignment is just a half of the entire course of action. Some instructors will request a copy of this report, others won't. Be certain to impress your scholars with an amazing work. If you are in possession of an intelligent action program, however, you might have the ability to clear a number of these hurdles to remain healthy and fit during the holiday season. It ought not sound too rehearsed. They're all available to download free of charge. They weren't pleased to see me. However, what if that committee was not actually a committee of individuals. Leave the hiring manager free of doubt you could do the job duties at the maximum level. The target of this sort of advertising is to remind clients and prospects of your brand along with the services and products you offer. If you don't have millions of dollars in your advertising budget, you have an extremely higher probability of failure with this sort of advertising. There's value within this bank of slow-to-mature prospects. Our prime concern and regions of discussion should now concentrate on how precisely the technology needs to be applied in individual households and in schools. There's no doubt this sort of marketing is effective, however, it's very expensive to successfully pull off and takes a great deal of time. Premium quality internet gambling home sites supply an eye-catching no deposit cash flow to entice gamers. In case it seems like a marketing spiel, folks have a tendency to switch off. However, a problem arises when small businesses attempt to imitate the huge brands at this form of marketing. College Essays - Part I - Common App Core Essay - Writing Eye Catching Essay: the Ultimate Convenience! She hadn't ever seen a complete solar eclipse before. And instead of trying hard to create your own from the bottom up, you just need to use these provided for you for download. Push harder than yesterday if you need a different tomorrow. Therefore, in the presentation design, there ought to be a call to action that gives clear next steps on how people are able to get in contact with you to choose the discussion forward. Capturing a person's attention in a brief span of time is a significant challenge. The other half contains paper formatting. The button is put below some exact clear bullet points describing what the site does, with an easy illustration near it so folks are absolutely clear what they're clicking for. Those who don't find time for exercise will need to find time for illness. Download free and conserve time. So How About College Essays - Part I - Common App Core Essay - Writing Eye Catching Essay? There are not any limits or restrictions on what you could do with it. This is a tough question to answer in absolute terms, because there is absolutely no strict right or wrong means to do it. Plagiarism is a critical matter that may create a lot of trouble. This will guarantee you keep your body energized while increasing your odds for success. Hustle to learn more muscle.

Electronic Marketing Essay Example | Topics and Well Written Essays - 2000 words

Electronic Marketing - Essay Example It would elaborate over how the advancements in technology and the advent of internet has assisted organisations in creating and improving customer value with the use of personalization and mass customization. Moreover, it would discuss the ethical and legal constraints associated with the use of internet. DISCUSSION The internet can be formally described as an interconnected network comprised of several networks and millions of computers that aim to link together businesses, educational institutions, government agencies, and individuals. There is no single organization controls the internet or how it functions, nor is it owned by any individual, yet it has provided the infrastructure for the transfer in commerce, scientific research and culture. The advent of internet has brought forward a plethora of opportunities and openings for businesses to take advantage of in order to better serve the customers. (Laudon & Traver, 2007) Marketing is basically the management process responsible for identifying, anticipating and satisfying the requirements of customers profitably with the help of the 4 Ps of the marketing mix, namely, product, price, promotion and place. (Baker, 2001) When internet and related technologies are used to communicate with the audience and the customers this becomes the process of electronic marketing. As the competition over the internet is increasing, there is more stress over providing the maximum value to the online customer. Customer value can be generated through providing them with a favourable experience. This experience can be enriched through the paradigms of internet accessibility, the depth and interactivity of the contents, faster access, convenience, speed of transactions and overall, a pleasant experience. All mass marketing comes down to offering customers with value for money. (Jones & George, 2007) Internet enables companies to produce customized products while maintaining a low unit cost for the products. Mass customization i s a new phenomenon that is taking over the business strategic environment. It can be defined as the enablement of a company to provided products that are tailored to the specific needs and demands of a group or individual bearing similar interest. The internet has increased the implementation of mass customization by companies. The youth of today are habituated to personalizing their experiences, be it the internet or their mobile phones. Customization is taking place over everything and anything, from toys to games to clothing. This generation is accustomed to personalization and customization from their brands and this is why more and more companies are going towards this phenomenon. The demands of the consumers are now less similar. They are unique and different from others making them less predictable for the companies. Mass customization allows companies to cater to specialized requests over a large scale. With the advent of globalization and the advancements in information tec hnology, companies are faced with greater competition from all around the globe. Every company is aiming to gain a presence over the internet which would make them ubiquitous and interactive. The Internet makes it possible, for companies large and small, to offer standardized and customized products and services compatibly, or to merely offer the latter. (Baylis et al, 2007) With the help of personalization, companies can target their marketing messages to individuals by

The Dos and Donts of Is It Proper to Write in the Second Person on a College Essay

The Do's and Don'ts of Is It Proper to Write in the Second Person on a College Essay The last paragraph should supply a well thought summary. Lastly, the detail of real speech makes the scene pop. You must be clear and specific with you statement in order for it to be understood. Don't forget to change up the verbiage used so you aren't simply copying your opening statement. The summary ought to be very different from the conventional summaries as it ought to be very solid. Then you may produce another draft. It can be difficult to choose what things to chat about in your college essays. Stephen's essay is quite effective. The rules for writing a great essay are the same. It's important to observe the conventional elements of an essay. Fortunately, you can learn how to craft a fantastic essay if you are able to stick to the typical pattern and write in a very clear and organized method. The subject for your very first paragragh ought to be in the initial and second sentence. When you compose an academic paper, bear in mind that the look of your paper can create a positive or negative impression on your reader. Essays aren't the only types of writing assignments you're very likely for. If you have to incorporate research, however, you are going to want to locate ideal examples from your sources. If you've completed any quantity of research, you will learn considerably more about a topic than you may conceivably include in a five or ten page essay. The MLA rule book states that the paragraphs should start with leaving a half-inch space at the start. You're presenting the sentence for a statement of fact rather than a personal belief. In the last paragraph, summarize what you've written. The very first paragraph might jump back and speak about the past. Is It Proper to Write in the Second Person on a College Essay Explained Each person encounters different struggles in regards to essay writing. To engage your reader and create your emotions and thoughts flow in the right fashion, you will need to understand some basic rules that are not the same as different essays. Obviously, essays play a major role in the college application procedure, also. The second-person presents an extremely different emotional connection with a slice of prose than either the initial or third manage. Whatever you decide to write about, you'll need to supply an essential judgement based on a set of criteria so it's vital to either pick a topic you know well or the one which you'll be in a position to research. It's very useful to take writing apart as a way to see just the way that it accomplishes its objectives. Without a transparent direction ahead, you're find it challenging to focus and keep relevant to the topic. Be certain that your outline makes perfect sense to avoid any issues during the writing process. You are the person making the judgement about this issue, it is all up to you to back up your argument in a sense that will make it possible for other people to create their own opinion. One means to do that's to work step-by-step, piece-by-piece. As an example, You perform better after a very good night's sleep'' uses the second-person perspective, although the idea may not apply to every reader. The very best thing to do is to use your good judgment, and you may always check with your instructor if you are uncertain of her or his perspective on the matter. Utilizing third-person writing can create a huge difference in giving your essay the ideal tone. There are various ways to grab a reader. You have to make the reader want to read whatever it's you've written. Rather than reading about somebody else's experiences, the reader of another person narrative is put into the story.

Human Carbonic Anhydrase II

Human Carbonic Anhydrase II Human carbonic anhydrase II is one of the fastest studied enzymes known with a variety of roles in reaction catalysis. Its primary function is to catalyze the reversible hydration reaction of carbon dioxide. In addition to carbon dioxide hydration, it is also capable of other latent skills, such as catalyzing esterase activity. The ability of human carbonic anhydrase II to function as a catalyst derives from key residues in and around the active site that play crucial roles in the mechanism. Substitutions to two of those particular key amino acids were performed via Quick-change site directed mutagenesis: H64A and V142D, to investigate the particular role they have in the catalytic active site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to discern and compare their activity to each other and to literature, including Michaelis-Menten parameters for PNPA hydrolysis, CO2 hydration, and inferring function molecular m odelling. Though the same trends can be seen as the literature, individual values were found to be much lower owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the active site: His64 is the proton shuttle that facilitates proton transfer during the rate limiting step and Val142 participates in the hydrophobic pocket to bind and recruit substrates to interact with the active site. Mutations to both of these sites show that enzyme efficiency and activity strongly decreases. Introduction Human carbonic anhydrase II (hCAII) is a zinc metalloenzyme that catalyzes the following reversible reaction: . The enzyme commonly functions to help shuttle carbon dioxide in red blood cells to rid the body of metabolic waste, and catalyzes the hydrolysis of many aromatic esters [1, 2]. Structurally, a zinc ion is located in the active site, coordinated to 3 histidine residues (H94, H96, H119) and usually a hydroxide ion or water molecule [2]. The mechanism of hCAII proceeds through two major steps: 1) the conversion of carbon dioxide to bicarbonate, and 2) the regeneration of Zn-OH by proton transfer. The active hydroxide that is bound to zinc nucleophilically attacks a nearby carbon dioxide molecule, resulting in a bicarbonate ion binding to zinc [3]. The zinc-oxygen bond breaks to subsequently release a bicarbonate ion, which is replaced with water [3]. The Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH bond is regenerated by a proton transfer to the external buffer, which is facilitated by the His64 residue [3]. The proton transfer step is the rate limiting step of the reaction [3]. The diazole side chain on the histidine residue is what gives it the ability to be a proton acceptor and donor. Mutations in that position (His64) usually result in decreased enzyme activity due to a lack of proton transfer; however the reaction does proceed to a lesser degree without an active His residue, possibly due to its extensive water network in the activ e site forming secondary proton wires [4]. Carbonic anhydrase catalyzes one of the most rapid reactions; it is one of the fastest enzymes studied [1]. Its reaction speed is due, in part, by the amphiphilic nature of the active site [1]. The hydrophobic side is used to bind carbon dioxide, while the hydrophilic patch functions to optimally orient the carbon dioxide molecule for the reaction [1]. The hydrophobic wall forms a well-defined pocket near the zinc-hydroxide and is composed of the following amino acids: Val142, Val121, Leu197 and Trp208. The hydrophilic patch consists of Thr198 and Glu106, which form a hydrogen bond network with the Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH to stabilize and orient it for nucleophilic attack on CO2 [2]. Therefore, any modifications to the hydrophobic pocket would change its structure, and consequently, its catalytic efficiency [1]. In this study, the importance and role of His64 and Val142 to the structure and mechanism of hCAII are determined through site-mutagenesis and subsequent characterization of the new mutants, H64A (His64 Æ’Â   Ala) and V142D (Val142 Æ’Â   Asp) via kinetic and structural analysis. The changes that arise from the substitutions may prove to be applicable to drug synthesis because hCAII is known to be involved in a variety of diseases, for example, Marble brain disease, where mutations in the hCAII gene leads to a deficiency in the enzyme which is an autosomal recessive disease [5]. Studies in hCAII mutations can be used to design folding modulators to suppress misfolding which frequently occurs due to hCAII destabilization [5]. Another major disease involved with hCAII gene is osteopetrosis. The hCAII genes inactivation decreases osteoclast function in bone, and knowledge of hCAII mutations that inactivate the enzyme may lead to better understanding of bone remodelling [6]. Some carbonic anhydrase diseases use inhibitors (CAI) to suppress the hCAII as a therapeutic treatment. Inhibitors prevent hCAII activity by inhibiting either of the reaction steps: the conversion of CO2 which involves V142 in the hydrophobic pocket, or the rate limiting step, proton transfer, in which His64 is crucial. Experimental Procedure Site directed mutagenesis via the PCR-based Quick-change method was performed on hCAII as cited in Woolley (2011) for 10 ng and 20 ng wild-type plasmids (hCA2pET24b from Novagen) [7]. Table shows the sequence of the primers used in the PCR reactions. Products of PCR mutagenesis reactions were run on 0.7% agrose gels to determine size. The gels were run at 150 V in 1X TAE buffer. Red safe dye from Intron Biotechnology was used in the agrose gel instead of ethidium bromide for safety reasons [7]. The standard molecular weight ruler used was a 1 kB DNA ladder from Fermentas. Table : Primer sequences used in mutagenesis of hCAII in the forward and reverse direction for mutants H64A and V142D Mutant Direction Sequence MW (Da) %GC TM ( °C) H64A Forward GGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG 10778 51 67 Reverse CTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC 10709 V142D Forward CTGATGGACTGGCCGaTCTAGGTATTTTTTTG 9868 44 62 Reverse CAAAAAAATACCTAGAtCGGCCAGTCCATCAG 9779 The enzyme, DpnI, was then used to digest methylated DNA (the parent template DNA). The DNA vector that contained the mutation was transformed into supercompetent E.coli turbo cells from New England Biolabs by heat shock [7]. LB-agar plates were prepared to grow the transformed cells containing mutant genes (i.e. H64A and V142D hCAII gene) [7]. Both were injected with Kanamycin to ensure that the culture that grows will have the desired mutation [7]. A miniprep culture was set-up from the LB-agar plate into LB medium to grow one colony for DNA analysis [7]. Restriction enzyme mapping was prepared and XhoI and BglII were chosen, they were used under buffer 3 for optimal efficiency. Plasmid putification was performed using the QIAprep Spin Miniprep Kit, and then the chosen restriction enzymes were carried out and were run on 1% agrose gel [7]. A sample of the purified DNA was sent to an external company (ACGT) for commercial sequencing (Sanger dideoxy type) to verify if the mutagenesis occurred correctly. The sequence was analyzed using the program BioEdit. To determine the level of confidence of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer [7]. The concentration was calculated using Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1(260) as 50 ÃŽÂ ¼g/mL. Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein expression by heat shock, similar to the transformation into turbo cells [7]. The cells were cultured and a single colony was grown. Once sufficiently grown, ITPG and ZnSO4 were added to induce protein expression [7]. SDS-PAGE was used to confirm protein expression and was analyzed against an unstained protein molecular weight marker by Fermentas. The protein and ladder was stained with coomassie blue [7]. Affinity chromatography was used to purify the mutant hCAII proteins [7]. The matrix used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight binding that occurs between hCAII and sulphonamides. Once purified, the protein was dialyzed using a 6000-8000 Da dialysis membrane to replace the elution buffer with protein buffer and removes the matrix from the protein [7]. SDS-PAGE is again used to confirm the protein is still present after purification and to check its approximate molecular weight. It was run for two different amounts of protein, 2 ÃŽÂ ¼g and 10 ÃŽÂ ¼g, and also ran 10ÃŽÂ ¼L of wash fractions from affinity chromatography [7]. Protein concentration was determined by UV absorption at 280 nm in a final concentration of 6M guanidine hydrochloride. From the calculated concentrations, purity of the protein could be assessed via SDS-PAGE. To characterize this purified hCAII protein, a variety of analyses were done. Two types of mass spectrometry (MS) were performed: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) [7]. The MS analysis was used to confirm the presence of the mutation in hCAII with intact and digested protein. Protein samples (H64A and V142D hCAII) were not diluted for either of the MS analyses as cited in Woolley (2010). Samples of 10 ÃŽÂ ¼L of stock protein concentrations (37.6 ÃŽÂ ¼M H64A and 3.2 ÃŽÂ ¼M V142D hCAII) were used for analysis of the molecular weight of the intact protein by ESI-MS. Both mutants were then digested by Trypsin Gold (MS grade) from Promega and the resulting fragments were evaluated by ESI-MS as well [7]. A 50 ÃŽÂ ¼L sample was used for each mutant, 40 ÃŽÂ ¼L of the mutant at stock concentration and 10 ÃŽÂ ¼L of the Trypsin Gold. A couple ÃŽÂ ¼L of the digested mutants were saved for MALDI-MS and the rest was used for ESI-MS. Once the molecular weights for each of the digested fragments were determined by ESI-MS, the products were run through a protein database to confirm the identity of the protein and mutations [7]. The 1 ÃŽÂ ¼L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 ÃŽÂ ¼L samples were mixed with a matrix consisting of 1 ÃŽÂ ¼L ÃŽÂ ±-cyano-4-hydroxycinnamic acid (CHCA) and 1 ÃŽÂ ¼L of 0.1% trifluoroacetic acid (TFA) [7]. The entire mixture was pipette onto a MALDI well and was inserted into the mass spectrometer and a MALDI-MS spectra was obtained. Michaelis-Menten kinetics was used to determine the KM and kcat of the p-nitrophenyl (PNPA) hydrolysis reaction [7]. The ionized product from the hydrolysis, p-nitrophenol (PNP) produces a bright yellow colour that was used to follow the rate of the reaction via the Perkin Elmer Lambda UV/Vis spectrophotometer [7]. Various sample concentrations of PNPA were set up to have a final enzyme concentration of 0.2 ÃŽÂ ¼M in protein buffer [7]. The initial rate measurements of each PNPA concentration were taken for wild-type enzyme, H64A mutant, V142D mutant, and a blank with no additional enzyme added (refer to data tables in Enzyme Kinetics I [7]). PNP has a molar absorption coefficient (ÃŽÂ µ) of 1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1. This was used to calculate Michaelis-Menten values: Vmax, KM, kcat, and kcat/KM [7]. The ability of hCAII mutants (H64A and V142D) and wild-type hCAII to catalyze the hydration of CO2 was measured. The pH of the solution was measured to track the progress of the reaction because the reaction generates protons. Enzyme solutions were prepared according to table 2 in [7]. The buffer used in the table was 50 mM TRIS buffer (pH 7.8). Additional enzyme samples were prepared for 25 nM of wild-type hCAII and 100 nM of H64A mutant in a final concentration of 22.5 and 29.92 mM imidazole buffer (pH 7.8) respectively to determine chemical rescue of mutant H64A. The pH of the CO2 hydration assay was measured using a pH probe and pH meter at 5 second increments for a total of 90 seconds starting at the beginning of the reaction [7]. The slope of the initial changes in the first 2 points was considered to be the V0 for each enzyme concentration. From the initial velocity, a kcat value can be calculated for each enzyme using the assumption that [S] >> KM, the Michaelis-Menten equati on simplifies to kcat=V0/[E]. The third kinetics experiment used fluorescence to determine the binding constant of dansyl amide (DNSA) and acetazolamide (AZ) (from Sigma-Aldrich) to H64A and wild-type hCAII was performed using the Perkin Elmer Fluorometer [7]. Stocks of 1 mM and 200 ÃŽÂ ¼M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 ÃŽÂ ¼M with TRIS buffer to make a 10 mL solution. A 1 mL sample of H64A from stock made was titrated with DNSA in small increments [7]. The fluorometer emissions were taken at 470 nm. AZ titration in competition with DNSA was not able to be completed. The last characterization experiment done was molecularly modelling the hCAII wild-type enzyme, as well as the mutants H64A and V142D. The molecular model of hCAII analyzed was derived by x-ray crystallography and found in the Protein Data Bank (PDB) repository. The wild-type and H64A hCAII structures examined had a PDB code of 1CA2 and 1MOO respectively. At present, no crystal structure has been found for V142D hCAII. The Swiss PDB Viewer program was used to visualize the protein structures. Secondary structures of the proteins were able to be observed. Residues around the metal active site and the Ramachandran plot were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB code 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic fit on the ÃŽÂ ±-carbons and structure alignment for each pair. The root mean square (RMS) between hCAII and the other isozymes were also analyzed to determine conserved and deviated regions in the structures. The binding of cobalt in the hCAII active site was also investigated (PDB code 3KOI). The structural inhibition of hCAII by AZ was also gleaned by structural analysis (PDB code 3HS4). Its mode of inhibition and binding sites were shown through the crystal structure. Lastly, the Swiss PDB Viewer program was used as a tool to theoretically synthesize mutations and compare it to the actual structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS. Results Site-directed mutagenesis PCR. Products from the PCR mutagenesis reactions were examined using 0.7% agrose gel electrophoresis. Two samples of differing amounts of template DNA (10 ng and 20 ng) were used for each mutant (Figure ). Bands were only observed for samples containing 20 ng of the hCA2pET24b DNA template plasmid (Figure ). The size of the bands observed coincides with the size of the plasmid used, 6018 bp. Heat shock transformation and isolation of plasmid. Several colonies were observed after plasmid transformation for both mutants, and 1 colony from each mutant was chosen for restriction enzyme digest with BglII and XhoI. Figure : Electrophoretic run on 0.7% agrose gel of DNA of hCAII mutants from PCR mutagenesis reactions. Lane 1 is the GeneRuler ladder by Fermentas and lanes 10-13 are the following: V142D (10 ng), V142D (20 ng), H64A (10 ng), and H64A (20 ng). As suggested from the gel, the mutants in the 20 ng plasmid was more successful than the 10 ng plasmids in determining relative molecular weights. Both mutants in the 20 ng plasmid show a band at approximately the 6000 base pair mark, which coincides with the number of base pairs in the hCA2pET24b plasmid that was used (6018 base pairs). Quantification of pure plasmid DNA. A 1/20th dilution was carried out on the purified DNA with elution buffer (EB; 0.1 M Tris, 0.4 M KSCN, pH 7). The absorption of the diluted DNA at 260 nm and 280 nm was taken by a UV/Vis spectrophotometer and the relative DNA purity was determined (Table ). The assumption that Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ). Table : Relative DNA purity for mutants V142D and H64A determined by UV/Vis spectrophotometer absorbance at 260 and 280 nm. Calculated concentrations of mutants from absorbance data, where Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL. Mutant Wavelength, ÃŽÂ » Absorbance Units Relative DNA Purity (A260/A280) Concentrated (ÃŽÂ ¼g/mL) Diluted (ÃŽÂ ¼g/mL) V142D 260 nm 0.3117 1.7852 311.70 15.59 280 nm 0.1746 H64A 260 nm 0.2653 1.7581 265.30 13.27 280 nm 0.1509 Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and together in a single and double digest for both mutants. The digested and undigested samples were run on 1% agrose gel, and 2 bands were observed around the 6000 and 7000 bp marker for all 8 samples (Figure , Figure ). The expected length of the bands in the double digest should be 892 bp and 5126 bp (Figure ). Figure : Electrophoresis performed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: V142D plasmid, V142D + XhoI, V142D + BglII + XhoI, and V142D BglII. Double bands are shown at the 6000 and 7000 bp marker for all 4 V142D samples. Figure : Electrophoresis performed in 1% agrose gel of digested H64A hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: H64A plasmid, H64A + XhoI, H64A + BglII + XhoI, and H64A BglII. Double bands are shown at the 6000 and75000 bp marker for all 4 H64A samples. Figure : Restriction enzyme cut sites and position of hCAII gene (5072-5854) on the hCAI2pET24b plasmid DNA Sequencing. The mutations for both V142D and H64A in the hCAII gene were successful according to the sequenced DNA result obtained from ACGT. Other mutations in the DNA sequence were observed in both mutants, but since the aligned protein sequence was the same, mutations were likely to be silent mutations due to amino acid redundancies. When sequenced in the forward direction by T7 polymerase, a protein mutation was found (K153N) other than the desired mutation of V142D; however, when sequenced in the reverse direction by T7 polymerase terminator (T7TER), K153N was not observed. Plasmid DNA transformation into E.Coli BL21(DE3) cells. Following transformation into BL21(DE3) cells, colonies were observed for both hCAII mutants (V142D and H64A). A random colony was chosen to be cultured and then was induced to express protein with 270 ÃŽÂ ¼M IPTG and 0.1 mM ZnSO4. SDS-PAGE for protein expression. Protein expression was tested with SDS-PAGE. The expected molecular weight of V142D hCAII is approximately 29.2 kDa and the expected molecular weight of H64A hCAII is approximately 29.1 kDa. SDS-PAGE bands are observed between the ladder markers 25.0 kDa and 35.0 kDa for both mutant proteins (Figure , Figure ). Figure : SDS-PAGE loaded with V142D hCAII proteins to examine protein expression. Samples were loaded in different volumes of protein to ensure gel visualization. Lane 15 contains the Fermentas protein molecular ladder and lane 1-4 contain the following: 1 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 4 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 1 ÃŽÂ ¼L+IPTG, 4 ÃŽÂ ¼L +IPTG. All 4 samples had some form of protein expression between 25.0 to 35.0 kDa. Figure : SDS-PAGE loaded with H64A hCAII protein to examine protein expression. One sample was loaded with 4 ÃŽÂ ¼L of H64A protein and +IPTG in lane 10. Lane 6 contains the Fermentas protein molecular ladder. The one H64A sample loaded showed an expression between 25.0 and 35.0 kDa. Calculation of pure protein concentration and extinction coefficient. Following affinity purification and dialysis, pure protein concentration was calculated from UV absorption measurements at 280 nm and the known extinction coefficient of hCAII as 50070 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3.2 ÃŽÂ ¼M and 37.6 ÃŽÂ ¼M respectively. Table : UV absorption measurements at 280 nm of purified protein and the resulting final concentration Mutant Average A280 Protein concentration (ÃŽÂ ¼M) V142D 0.01583 3.2 H64A 0.1884 37.6 SDS-PAGE to assay purity and check approximate molecular weight. Several samples were loaded into the SDS-PAGE for each mutant protein: lysate and wash fractions (collected from affinity chromatography), 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. For H64A, a visible band was only observed for the 10 ÃŽÂ ¼g sample (Figure ). The band was located between the 35 kDa and 25 kDa markers on the ladder. For V142D, none of the 4 samples resulted in a band on the gel (Figure ). Figure : SDS-PAGE shown for H64A mutant protein. Lane 1 contains the Fermentas protein molecular weight marker. Lane 11-14 contains H64A samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. Only the 10 ÃŽÂ ¼g protein had (faint) observable bands located between the 25 and 35 kDa markers. Figure : SDS-PAGE shown for V142D mutant protein. Lane 4 contains the Fermentas protein molecular weight marker. Lane 12-15 contains V142D samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. No observable bands are seen for any of the samples. Mass spectrometry. ESI-MS was not successful in analyzing the molecular weight of intact and digested protein of both mutants. A MALDI spectrum was able to be generated for the digested proteins; however, without the digested ESI spectrum to compare to, the peaks from the MALDI spectrum can only be speculatively assigned. Kinetics: Hydrolysis of PNPA. Using the molar absorption coefficient of PNP (1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), the rate of each reaction was determined. The predicted rate was calculated using the Michaelis-Menten kinetics: . The plot of predicted rates and actual initial rates vs. PNPA concentration can be seen in Figure , Figure , Figure for wild-type, H64A, and V142D hCAII respectively. The Vmax and KM values for each enzyme were calculated by minimizing the square difference between the predicted and actual reaction rates, and the kcat was calculated using the equation: (Table ). Table : Calculated Michaelis-Menten parameters for wild-type, H64A, and V142D hCAII catalyzing the hydrolysis of PNPA. Wild-type hCAII H64A hCAII V142D hCAII Vmax (ÃŽÂ ¼M/sec) 1.202 0.812 0.218 KM (mM) 1.280 1.957 8.362 kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.141 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢3 2.159 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 6.825 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 kcat/KM (Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 4.02 11.032 8.162 Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for wild-type hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for H64A hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for V142D hCAII enzyme. Kinetics: CO2 hydration. Initial velocity (V0) values were calculated by measuring the progression of the reaction (via concentration of protons) with time (Table , Table , and Table ). kcat values were then calculated using the same equation as in the hydration of PNPA and averaged for the individual enzymes (wildtype, H64A, and V142D hCAII) in a particular buffer (i.e. TRIS or imidazole). Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by wild-type hCAII in TRIS buffer and imidazole buffer. Wild-type concentration (nM) V0 for WT+TRIS (M/s) V0 for WT+Imidazole (M/s) 0 1.3E-08 6.05778E-08 1.5 1.1E-08 N/A 2.5 1.1E-08 5.63E-08 5 2.1E-08 5.16E-08 12.5 5.9E-08 5.63E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.3 ±1.62 12.44 ±9.19 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by H64A hCAII in TRIS buffer and imidazole buffer. H64A concentration (nM) V0 for H64A+TRIS (M/s) V0 for H64A+Imidazole (M/s) 12.5 1.4E-08 6.57E-08 25 1.4E-08 5.8E-08 50 1.7E-08 7.53E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.67 ±0.39 3.03 ±1.97 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by V142D hCAII in TRIS buffer. V142D concentration (nM) V0 for V142D+TRIS (M/s) 12.5 6.2E-09 25 5.4E-09 50 5.5E-09 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.27 ±0.19 Fluorescence detection of ligand binding. DNSA was titrated with H64A hCAII to determine its affinity for the enzyme. The dissociation constant, KD, for DNSA was determined to be 0.086 ÃŽÂ ¼M when protein concentration was 0.25 ÃŽÂ ¼M. Competitive titration of H64A-DNSA hCAII with AZ was attempted, but was not successful as DNSA binding was too tight, making it difficult to be displaced by AZ. Molecular modeling. Literature models of wild-type (PDB code 1CA2) and H64A (PDB code 1MOO) hCAII were analyzed. There is no available structure of V142D hCAII at present. The secondary structure of wild-type is composed of 18 ÃŽÂ ²-sheets (77 residues) and 10 ÃŽÂ ±-helices (42 residues), with the majority of the ÃŽÂ ±-helices falling in the domain of right-handed helices, while very few show left-handed helical properties according to the Ramachadran plot. It also seems that the active site is solely composed of ÃŽÂ ²-sheets, and no ÃŽÂ ±-helices (Figure ). Analyzing PDB structure 3HS4 (AZ bound hCAII), the mechanism as to how AZ inhibits hCAII function can be seen. AZ has 3 binding sites, 2 are novel binding sites and the other provides a mechanism of inhibition. AZ binds the zinc directly at the active site, displacing crucial ligands needed for catalysis. There were some discrepancies found between the crystal structure of H64A [1MOO] as cited on PDB and virtually mutated H64A from wild-type hCAII, resulting in a RMSD (root mean square deviation) of 0.29 Ã… (Figure ). Since no literature structure of V142D is available, no comparison between virtual and crystal structures could be made. Figure : Secondary structure of wild-type hCAII overlain with ribbon to visualize the higher arrangement. Figure : RMSD between H64A hCAII virtually mutated and literature crystal structure. Blues denote the same or similar residues, while reds and oranges indicate completely different amino acids. Discussion Agrose gel results were only visible for samples that contained 20 ng of the plasmid template DNA, rather than the 10 ng plasmid. This may be a result of more amplification during PCR with the 20 ng plasmid, and so would intensely be more visible. Though the 20 ng samples showed bands at the appropriate 6000 bp mark, there was also a faint band that can be seen near the end of the gel. This may be due to non-specific primer annealing. Quantification of DNA purity was done by exploiting the peak absorbances of protein and DNA. DNA maximally absorbs at 260 nm, while protein dominantly absorbs at 280 nm. The purity ratio reports the relative amount of DNA compared to protein present in the sample. The purity of both mutants were approximately 1.8, which is regarded as a relatively pure sample; however, a purity ratio of more than 2.0 would have been ideal. The restriction enzyme digest showed 2 bands (7000, 6000 bp) for all samples, which may have been a sign of poor mixing/ pipetting since the volumes of restriction enzyme were extremely small amounts. If this is the case, only some of the DNA was nicked and some were not, which would result in 2 bands. It was expected that the plasmid sample would have a high band (supercoiled), each of the singly digested samples would have a slightly lower band (nicked), and the doubly digested would show 2 bands that indicated the fragment size of 892 and 5126 bp. Sequencing results showed that a protein mutation occurred when the sample was sequenced in the forward direction by the T7 polymerase. A lysine at position 153 had mutated to glutamine (K153N). However, this mutation was not observed when the T7 polymerase terminator was used to sequence the sample in the reverse direction. A mutation that occurs in one sequencing direction and not the other is usually attributed to sequencing errors, which may be the reason in this case. The SDS-PAGE bands for protein expression coincided with the expected molecular weight for both mutants, which could suggest that the correct proteins were expressed; however, there is a possibility that the proteins expressed could be of similar weight, but completely different. Interestingly, the V142D samples that did not include the protein inducer, IPTG, had a more intense band than the faint ones found for the samples that did include IPTG. This may just be a result of mislabelling. The SDS-PAGE performed to assess purity after the purification process. Mutant V142D had low protein expression as evidenced by its concentration of 3.2 ÃŽÂ ¼M. The V142D mutant should have very low protein expression according to Fierke et al. (1991) because valine at position 142 is uniquely required for maximal expression in E.Coli. It is suggested that by altering position 142, protein stability decreases [2]. Therefore, the protein that was expressed in the previous SDS-PAGE gel may not be V142D hCAII at all. The sample may have been small fragmented contaminant proteins that would have completely run off the gel altogether. However, the low concentration of V142D after purification may also be a major factor in the lack of gel bands observed as well. Unlike V142D, H64A hCAII concentration should not have affected its lack of bands because it was calculated to have had a reasonable concentration of 37.6 ÃŽÂ ¼M. There were some problems loading the samples into the wells; t his could be an explanation as to no observable gel bands. ESI-MS is dependent on concentration because it affects the size of primary droplets [8]. The unsuccessful determination of molecular weight of V142D hCAII may be attributed to its low concentration. The H64 hCAII mutant was also not able to be successfully analyzed with ESI-MS. A possible reason for the failure was that it was not kept on ice while it was not being used. The enzyme may have become inactive and degraded into smaller fragments. This would explain the ESI-MS output obtained for H64A. No definite molecular mass was determined, but the spectrometer did detect a lot of small protein fragments in the sample, all under 1000 amu. The kinetic values obtained from PNPA hydrolysis do not follow similar trends found in literature [2]. The kcat/KM for wild-type hCAII (2500 ±200 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) was found to be significantly larger than V142D hCAII (3 ±0.3 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) in literature, more than 800ÃÆ'- larger [2]. Experimental calculations yielded kcat/KM for V142D (8.16 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) to be about 2ÃÆ'- larger than wild-type (4.02 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), which did not follow literature patterns. The literature trends make more biolog