Chapter 1 Laboratory Techniques 09 09 2015 Laboratory Safety Equipment Eyewash Fire Extinguisher First Aid Kit Safety Glasses Gloves Laboratory Safety Procedures Do not eat drink or smoke in lab Always wash hands prior to leaving the lab Always wear closed toe shoes NO SANDALS Types of Pipets Mechanical pipets Micropipets less than 1 mL are used for small volume transfers precisely Mohr Volumetric Transfer Pipet are designed to deliver a single volume is a measuring pipet o Pipet is graduated but stops at a baseline before the pipet begins to narrow o The meniscus must be precisely on a calibration ark both at the beginning and at the end of a transfer o The marking near the meniscus is the amount you want to put into the mixture Serological Pipet are graduated to deliver there is no baseline o The entire amount measured will be transferred for this type of pipet o Serological pipets are labeled with the zero mark at the top of the pipet you will need to subtract the amount you are going to pipet from the total volume of the pipet to determine the exact mark to fill the pipet to Always use pi pumps for filling pipets o A pi pump is a device designed to safely and easily draw fluid up into a pipet Spectrophotometry o A spec is an instrument designed to detect the amount of radiant energy absorbed by the molecules o The concentration of a substance in solution increases the amount of light absorbed by the solution increases o The spec has 2 different scales Transmittance measured from 0 100 Absorbency Measured from 0 2 o All solutions of chemical compounds absorb light of specific wavelengths therefore the spec can be useful in identifying compounds o Steps for using Spec Turn on Spec an allow to warm up for 15 mins Adjust wavelength to appropriate value With the sample holder EMPTY and lid closed adjust the Zero Adjust Knob until the instrument read 0 on transmittance scale Then put the blank in and close lid Adjust the 100 Adjust Knob on the right front of instrument to read 100 transmittance Remove the blank immediately and insert the sample and then switch the mode to read to absorbency Chapter 2 Analyzing Enzymes and Enzyme Kinetics 09 09 2015 Background Info Catalysts Increases the rate of a chemical reaction without being consumed or changed during the process This change in the reaction rate is accomplished by reducing the Activation Energy between reactants and products The chemical reactions that are part of like are made possible by organic biological catalysts known as enzymes o Unlike other catalysts enzymes are highly specific Most enzymes are protein and therefore have complex 3 dimensional shape or conformations o The conformation of an enzyme is linked to its function A general model of enzyme catalysis begins with the enzyme and substrate reactant molecules separate The enzyme binds the substrate and they form the enzyme substrate complex After this the reaction can take place The result of the reaction converts the substrate into the product and the product will detach from the enzyme since it no longer fits o A specific example of this Induced Fit model FIG Active Site The substrate is attracted to this site and this is where the reaction takes place Exercise 1 The enzyme is an oxidase and because catechol is the substance being oxidized a more exact name for the enzyme is catechol oxidase aka catecholase Reactants Catechol and Oxygen are converted to Benzoquinone on We are using potatoes in this experiment and measuring the rate the enzyme catecholase they brown Benzoquinone brown on fruit In this reaction we are looking at the rate of the browning of potatoes and we can do this using the spec The more benzoquinone more brown there will be the more absorbency there will be Measuring o How quickly the reactants are being taken up o How quickly the product is being produced Blank dH2O Results o The results showed that has time progressed the absorbency increased because there was more browning and therefore so did the concentration Alternative Hypothesis Scientific Method Null Hypothesis is a method to logically answer questions states that the experiment will show no difference Exercise 2 in the results between control group and the experimental group alternative to the null and states that there always is a difference between the control and experimental group In science you can only disprove never PROVE something to its true value describes the center of the population Standard Deviation the points spread refers to the closeness of a measure of calculated value is the closeness of repeated measurement to one another The mean is equal to the average value and as such This describes how far away from the center Precision Mean Accuracy Using both standard deviation and mean together the investigators can compare two or more population of data This experiment did several different treatments o Enzyme Concentration Need a new blank for each new concentration of catecholase o pH o Substrate Concentration Also need a new blank for each new concentration Only changing catechol and oxygen not enzyme so don t need a new blank o Temperature Do not need a new blank o Structure of the substrate enzyme o Source of Enzyme Do not need a new blank because we are not changing Changing enzyme so need a new blank If you change anything that deals with the enzyme you have to blank it Most blanks have everything in them that s in the experiment minus what you are changing except this experiment because our blank would react so it is just dH2O Chapter 3 Movement of Materials Across the Cell Membrane 09 09 2015 Background Info Membranes allow cells an organisms to control their internal chemical environment The cell membrane is selectively permeable The ability of a molecule to cross biological membrane is a function of many factors including o Size of molecule o Charge o Hydrophobic hydrophilic character o Concentration Hydrophobic refers to substances that are lipid soluble Hydrophilic refers to substances which dissolve easily in water Very small uncharged molecules can pass directly through the phospholipid bilayer Phospholipid bilayer o Hydrophilic heads o Hydrophobic tails If water molecule can move to sites of lower concentration across membrane then there will be a decrease in organization or free energy The change in free energy of the water molecules is based on the following equation o G H T S G the change in free energy H the change in enthalpy heat energy T temperature