KINETICS OF A SINGLE SUBSTRATE ENZYME-CATALYSED REACTION; THE HYDROLYSIS OF P-NITROPHENYL PHOSPHATE BY MUCOSAL ALKALINE PHOSPHATASE
Title of lab repeat: KINETICS OF A SINGLE SUBSTRATE ENZYME-CATALYSED REACTION; THE HYDROLYSIS OF P-NITROPHENYL PHOSPHATE BY MUCOSAL ALKALINE PHOSPHATASE Instructions: 1. This practical should be written in the format of a research paper which you are planning to submit to the Biochemical Journal. 2. Look up the instructions to authors, this will tell you in what format you will produce the write up in. The following links are useful http://www.biochemj.org/content/submission-guidelines. This gives access to the website of Portland Press the publishers of the Biochemical Journal. Also look at some of the actual journal research articles to have an idea of how the paper should be produced/look like. 3. The calculations for the, gradients (slopes of curves), Vo, Vmax and km need to be very clearly shown. 4. The graphs to be included in the paper need to be hand drawn clearly, please try to make them presentable and not messy then scan them and insert into your document, not Excel produced. [unique_solution]Make sure the legend for each graph is well written and has enough information to explain what it is. 5. Make sure that all parts of the report are clearly shown, i.e. title page with author name and affiliation, abstract, introduction, materials and methods, results, discussion and reference sections. 6. As you have been informed at the beginning of the term; a. If you have not attended all lab sessions (unless you had a genuine reason to which an extenuation circumstances form supported with evidence has been submitted and accepted), and b. If the paper is submitted in any other format ………… it will not be marked. For the calculations; 1. You will initially produces 2 graphs for the time courses one without inhibitor and one with inhibitor. 2. These will be used to determine Vo which is the slope of each line on the graph (at different substrate concentration). Graphs are Abs reading vs. time in seconds. 3. The next graph will be the Michellis-Menton graph this is Vo vs. substrate concentration. Here you will produce ONE graph with two lines +/- inhibitor. 4. The final graph will be the double reciprocal (Lineweaver Burk plot), the following link will be useful, https://www.youtube.com/watch?v＝5upkDCe-v8k. 5. Then using the instructions in your practical protocol calculate the Ki for the inhibitor. 6. So you need to show the following; a. initial velocity, Vo, for each different substrate concentration +/- inhibitor b. maximal velocity, Vmax, for the enzyme system +/- inhibitor. c. Km for the enzyme system +/- inhibitor. d. And finally the Ki for the inhibitor.