1 00:00:00,000 --> 00:00:02,425 [SQUEAKING] 2 00:00:02,425 --> 00:00:03,395 [RUSTLING] 3 00:00:03,395 --> 00:00:05,335 [CLICKING] 4 00:00:14,545 --> 00:00:15,920 SARAH HEWETT: Today, we are going 5 00:00:15,920 --> 00:00:19,920 to talk about the second part of the catalase lab. 6 00:00:19,920 --> 00:00:21,990 So, in the first part of the catalase, 7 00:00:21,990 --> 00:00:24,500 we had catalase and hydrogen peroxide. 8 00:00:24,500 --> 00:00:27,980 And we were measuring the rate of the decomposition 9 00:00:27,980 --> 00:00:29,450 of hydrogen peroxide as catalyzed 10 00:00:29,450 --> 00:00:30,962 by the catalase enzyme. 11 00:00:30,962 --> 00:00:32,420 And we were doing that by measuring 12 00:00:32,420 --> 00:00:33,740 the pressure of oxygen. 13 00:00:33,740 --> 00:00:36,032 And, for the people who are currently 14 00:00:36,032 --> 00:00:38,240 doing the catalase lab, you've already done part one. 15 00:00:38,240 --> 00:00:39,698 And, for everybody else, you'll get 16 00:00:39,698 --> 00:00:42,740 to experience that coming up in the next lab or two. 17 00:00:42,740 --> 00:00:44,750 And then, in part two of the catalase lab, 18 00:00:44,750 --> 00:00:47,352 we are going to look at quantifying 19 00:00:47,352 --> 00:00:49,310 the amount of catalase that we have in a sample 20 00:00:49,310 --> 00:00:52,460 and then looking at the iron centers in the catalase 21 00:00:52,460 --> 00:00:55,530 and trying to quantify those as well. 22 00:00:55,530 --> 00:00:58,430 So we are going to go back to the beginning 23 00:00:58,430 --> 00:01:02,010 and talk a little bit more about proteins and protein structure. 24 00:01:02,010 --> 00:01:04,018 So, if you have taken a biology course 25 00:01:04,018 --> 00:01:06,560 at any point in your life, then this will probably be review. 26 00:01:06,560 --> 00:01:08,435 But, just so that we're all on the same page, 27 00:01:08,435 --> 00:01:11,900 we want to go back and say that the building blocks of proteins 28 00:01:11,900 --> 00:01:13,290 are amino acids. 29 00:01:13,290 --> 00:01:15,840 And this is the general structure of an amino acid. 30 00:01:15,840 --> 00:01:17,510 And, if you change this R group here 31 00:01:17,510 --> 00:01:21,648 to any one of these R groups over in this table, 32 00:01:21,648 --> 00:01:23,690 then you can get all of the different amino acids 33 00:01:23,690 --> 00:01:26,300 that are naturally produced in nature. 34 00:01:26,300 --> 00:01:29,120 And there are 20 amino acids that occur in nature. 35 00:01:29,120 --> 00:01:31,400 And they vary by these R groups. 36 00:01:31,400 --> 00:01:32,900 And, depending on the R group, there 37 00:01:32,900 --> 00:01:35,740 are different classes of amino acids that you can have. 38 00:01:35,740 --> 00:01:37,850 So there are the nonpolar ones where 39 00:01:37,850 --> 00:01:40,190 you have an alkyl or an aromatic side chain. 40 00:01:40,190 --> 00:01:42,530 You can have polar side groups where 41 00:01:42,530 --> 00:01:48,320 you have a hydroxyl group or a thiol or some amine groups. 42 00:01:48,320 --> 00:01:50,210 And then you can have acidic or basic groups 43 00:01:50,210 --> 00:01:51,460 that are electrically charged. 44 00:01:51,460 --> 00:01:53,127 So, depending on the pH of the solution, 45 00:01:53,127 --> 00:01:54,630 they'll either be protonated or not. 46 00:01:54,630 --> 00:01:56,797 And then you can have positive and negative charges. 47 00:01:56,797 --> 00:01:59,750 And all of those contribute to the protein structure 48 00:01:59,750 --> 00:02:02,750 and the properties of the protein, what it can do. 49 00:02:02,750 --> 00:02:04,553 And just a review of terminology, 50 00:02:04,553 --> 00:02:06,470 so proteins are made of chains of amino acids. 51 00:02:06,470 --> 00:02:09,590 A peptide is two or more amino acids stuck together. 52 00:02:09,590 --> 00:02:13,210 A polypeptide is when you get 10 or more amino acids stuck 53 00:02:13,210 --> 00:02:13,710 together. 54 00:02:13,710 --> 00:02:15,758 And then, if you have beyond that, 55 00:02:15,758 --> 00:02:18,050 once it gets to longer and longer chains of amino acids 56 00:02:18,050 --> 00:02:19,550 and they start folding up and having 57 00:02:19,550 --> 00:02:22,142 specific functions, that's when it becomes a protein. 58 00:02:22,142 --> 00:02:23,600 So, if you hear those terms, that's 59 00:02:23,600 --> 00:02:25,830 what we're talking about. 60 00:02:25,830 --> 00:02:28,970 So the first-- the most basic structure 61 00:02:28,970 --> 00:02:31,560 that you can have in a protein is the primary structure. 62 00:02:31,560 --> 00:02:33,778 And that is just the sequence of amino acids. 63 00:02:33,778 --> 00:02:35,570 So you have this long chain of amino acids. 64 00:02:35,570 --> 00:02:36,620 You stick them together. 65 00:02:36,620 --> 00:02:38,220 And that is the primary structure. 66 00:02:38,220 --> 00:02:40,490 So, if this is our little peptide chain here, 67 00:02:40,490 --> 00:02:43,280 we have alanine, threonine, tyrosine, and valine. 68 00:02:43,280 --> 00:02:46,707 And those are from these side groups is how 69 00:02:46,707 --> 00:02:48,650 you know what amino acid it is. 70 00:02:48,650 --> 00:02:51,590 And then the bond in between the amino acids here 71 00:02:51,590 --> 00:02:54,020 where your carbonyl group of one amino acid bonds 72 00:02:54,020 --> 00:02:56,300 to the amine group of another amino acid, that 73 00:02:56,300 --> 00:02:57,842 is your peptide bond. 74 00:02:57,842 --> 00:02:59,300 And so you can get chains of these, 75 00:02:59,300 --> 00:03:02,180 and the primary structure again is just the list 76 00:03:02,180 --> 00:03:04,550 of amino acids in a line. 77 00:03:04,550 --> 00:03:07,400 That's about as simple as it gets. 78 00:03:07,400 --> 00:03:10,340 The secondary structure is how the amino acids interact 79 00:03:10,340 --> 00:03:14,756 with one another to form 3D structures 80 00:03:14,756 --> 00:03:16,830 within the polypeptide chain. 81 00:03:16,830 --> 00:03:18,785 So, if you have two chains of amino acids 82 00:03:18,785 --> 00:03:20,660 or if you have one that's folded onto itself, 83 00:03:20,660 --> 00:03:23,115 it can form hydrogen bonds between these oxygens 84 00:03:23,115 --> 00:03:24,740 of the carbonyls and the hydrogens that 85 00:03:24,740 --> 00:03:28,070 are attached to the nitrogens in the protein backbone. 86 00:03:28,070 --> 00:03:29,480 And you can get these beta sheets 87 00:03:29,480 --> 00:03:33,470 that are kind of pleated if you look at them in actual 3D 88 00:03:33,470 --> 00:03:34,670 space. 89 00:03:34,670 --> 00:03:37,340 And you can have one long chain that bonds 90 00:03:37,340 --> 00:03:40,160 to itself in the similar fashion with the hydrogen bonding. 91 00:03:40,160 --> 00:03:42,080 And you can get alpha helices. 92 00:03:42,080 --> 00:03:45,050 And it creates a helical shape within the protein backbone. 93 00:03:45,050 --> 00:03:47,612 And these are the main types of secondary structures 94 00:03:47,612 --> 00:03:48,320 that you can get. 95 00:03:48,320 --> 00:03:50,028 There are other ways that the protein can 96 00:03:50,028 --> 00:03:52,760 interact with itself and form different structures, 97 00:03:52,760 --> 00:03:55,640 but these are the major two. 98 00:03:55,640 --> 00:03:57,920 Structures are held together using hydrogen bonds. 99 00:03:57,920 --> 00:04:02,437 And then you can get tertiary structure. 100 00:04:02,437 --> 00:04:04,520 So there are many different ways that proteins are 101 00:04:04,520 --> 00:04:06,870 represented in graphical form. 102 00:04:06,870 --> 00:04:07,970 So you can have-- 103 00:04:07,970 --> 00:04:10,993 you've seen in our past lecture with the catalase, 104 00:04:10,993 --> 00:04:13,160 you can have a space-filling model where all of it's 105 00:04:13,160 --> 00:04:14,840 kind of like the ball and stick where it just 106 00:04:14,840 --> 00:04:15,980 looks like one giant blob. 107 00:04:15,980 --> 00:04:18,920 And that can give you a sense of the overall shape 108 00:04:18,920 --> 00:04:20,390 of the protein. 109 00:04:20,390 --> 00:04:22,010 You can also have line structures 110 00:04:22,010 --> 00:04:23,120 where it's kind of like the line structure 111 00:04:23,120 --> 00:04:24,350 that you would draw in organic chemistry 112 00:04:24,350 --> 00:04:25,670 where it shows all of the different side 113 00:04:25,670 --> 00:04:26,840 chains in the backbone. 114 00:04:26,840 --> 00:04:29,210 And then you have these ribbon structures 115 00:04:29,210 --> 00:04:31,310 where you can see sort of the alpha helices 116 00:04:31,310 --> 00:04:32,900 and maybe some beta sheets over here. 117 00:04:32,900 --> 00:04:35,150 And it shows some different features 118 00:04:35,150 --> 00:04:37,190 of the structure of a protein. 119 00:04:37,190 --> 00:04:39,620 But the tertiary structure is how 120 00:04:39,620 --> 00:04:41,930 all of the elements of the secondary structure 121 00:04:41,930 --> 00:04:44,850 interact to form the overall shape of the protein. 122 00:04:44,850 --> 00:04:47,250 And it can be formed by various interactions. 123 00:04:47,250 --> 00:04:49,850 And so, while the secondary structure is mostly 124 00:04:49,850 --> 00:04:55,680 formed by interactions between the backbone functional groups, 125 00:04:55,680 --> 00:04:58,010 the, tertiary structure comes from the interactions 126 00:04:58,010 --> 00:04:59,010 between the side chains. 127 00:04:59,010 --> 00:05:00,927 So you can have these hydrophobic interactions 128 00:05:00,927 --> 00:05:03,950 if you have alkyl or aromatic side chains. 129 00:05:03,950 --> 00:05:06,630 So, if your protein is in a cell, 130 00:05:06,630 --> 00:05:08,850 then it is in a mostly aqueous environment. 131 00:05:08,850 --> 00:05:10,320 And aqueous environments are polar. 132 00:05:10,320 --> 00:05:12,450 And so these nonpolar groups will 133 00:05:12,450 --> 00:05:14,790 tend to coagulate together. 134 00:05:14,790 --> 00:05:16,090 You can have disulfide bonds. 135 00:05:16,090 --> 00:05:20,040 So, if you have the protein side chains that have sulfur groups, 136 00:05:20,040 --> 00:05:23,680 then they can make covalent bonds between the sulfurs. 137 00:05:23,680 --> 00:05:24,930 You can have hydrogen bonding. 138 00:05:24,930 --> 00:05:28,380 Again, those different side chains 139 00:05:28,380 --> 00:05:31,230 have again hydrogens that are attached 140 00:05:31,230 --> 00:05:33,990 to nitrogens and oxygens and carbonyl groups 141 00:05:33,990 --> 00:05:35,370 that can form hydrogen bonds. 142 00:05:35,370 --> 00:05:38,160 And then those electrically charged side chains 143 00:05:38,160 --> 00:05:39,130 can form salt bridges. 144 00:05:39,130 --> 00:05:40,630 So, if you have a positively charged 145 00:05:40,630 --> 00:05:43,110 and a negatively charged amino acid near each other, 146 00:05:43,110 --> 00:05:46,222 then they can have an ionic interaction like that. 147 00:05:46,222 --> 00:05:48,180 Interactions with the solvent also play a role. 148 00:05:48,180 --> 00:05:50,210 And this is what holds the protein together 149 00:05:50,210 --> 00:05:54,110 in its functional shape. 150 00:05:54,110 --> 00:05:56,990 And then, lastly, we have our quaternary structure. 151 00:05:56,990 --> 00:05:58,820 And this is formed by the interaction 152 00:05:58,820 --> 00:06:00,440 of multiple subunits coming together 153 00:06:00,440 --> 00:06:03,050 to make a larger protein. 154 00:06:03,050 --> 00:06:05,900 So this is a picture of catalase. 155 00:06:05,900 --> 00:06:09,195 And you can see each of the subunits of the catalase 156 00:06:09,195 --> 00:06:10,070 is a different color. 157 00:06:10,070 --> 00:06:12,398 So catalase has four different subunits. 158 00:06:12,398 --> 00:06:14,690 So these four subunits all have their own secondary and 159 00:06:14,690 --> 00:06:15,482 tertiary structure. 160 00:06:15,482 --> 00:06:16,530 They're identical. 161 00:06:16,530 --> 00:06:19,100 And then they come together to make the overall catalase 162 00:06:19,100 --> 00:06:20,188 protein. 163 00:06:20,188 --> 00:06:21,980 Not all proteins have quaternary structure. 164 00:06:21,980 --> 00:06:25,790 Some just have normal folding and secondary and tertiary 165 00:06:25,790 --> 00:06:27,435 structure. 166 00:06:27,435 --> 00:06:29,810 And the quaternary structures are typically held together 167 00:06:29,810 --> 00:06:32,030 by dispersion forces and hydrogen bonding 168 00:06:32,030 --> 00:06:33,300 between the subunits. 169 00:06:33,300 --> 00:06:35,948 So it's not usually a covalent interaction, 170 00:06:35,948 --> 00:06:38,240 but it is strong enough to hold these subunits together 171 00:06:38,240 --> 00:06:41,650 into the correct shape for the protein to function. 172 00:06:44,490 --> 00:06:47,580 Another key piece of protein structure 173 00:06:47,580 --> 00:06:48,910 are prosthetic groups. 174 00:06:48,910 --> 00:06:51,780 And these are groups that are not made out of amino acids. 175 00:06:51,780 --> 00:06:54,630 So they are not peptides, but they are very 176 00:06:54,630 --> 00:06:57,090 important for protein function. 177 00:06:57,090 --> 00:06:59,520 And some examples are iron-sulfur clusters. 178 00:06:59,520 --> 00:07:01,920 So these are some 3D representations 179 00:07:01,920 --> 00:07:03,420 of what an iron-sulfur cluster would 180 00:07:03,420 --> 00:07:06,150 look like where your iron centers are red, 181 00:07:06,150 --> 00:07:08,640 and the sulfur are these yellow balls. 182 00:07:08,640 --> 00:07:10,980 So you can have different numbers of irons and sulfurs 183 00:07:10,980 --> 00:07:12,030 bonded together. 184 00:07:12,030 --> 00:07:14,910 And these play key roles in photosynthesis 185 00:07:14,910 --> 00:07:16,420 and in metabolism. 186 00:07:16,420 --> 00:07:19,250 So they're really good at electron transport. 187 00:07:19,250 --> 00:07:20,730 So, in the electron transport chain 188 00:07:20,730 --> 00:07:23,835 in your cellular metabolism or in photosynthesis 189 00:07:23,835 --> 00:07:25,710 when you have to transport electrons in order 190 00:07:25,710 --> 00:07:30,030 to make energy, those iron-sulfur clusters, 191 00:07:30,030 --> 00:07:33,310 that is where they are typically found in nature. 192 00:07:33,310 --> 00:07:35,230 And another really common prosthetic group 193 00:07:35,230 --> 00:07:39,100 is the heme group, which you may recognize from hemoglobin. 194 00:07:39,100 --> 00:07:41,760 It is used in oxygen binding and transport. 195 00:07:41,760 --> 00:07:44,440 So it binds to the oxygen and carries it through your blood. 196 00:07:44,440 --> 00:07:48,610 And it can also be used in oxygen reactions or oxygen 197 00:07:48,610 --> 00:07:51,490 activation, which is what we're going to talk about today. 198 00:07:51,490 --> 00:07:53,140 Other types of prosthetic groups, 199 00:07:53,140 --> 00:07:55,007 you can have lipids or sugars that 200 00:07:55,007 --> 00:07:57,340 are bound to proteins that help with protein recognition 201 00:07:57,340 --> 00:08:00,760 and things like that, but metals and metals centers 202 00:08:00,760 --> 00:08:05,063 are the most common that are used. 203 00:08:05,063 --> 00:08:06,730 So, if we want to talk a little bit more 204 00:08:06,730 --> 00:08:08,470 about the heme group is what we're going to focus on, 205 00:08:08,470 --> 00:08:11,240 the heme group is based off of this porphyrin structure. 206 00:08:11,240 --> 00:08:13,060 And so this is a porphyrin. 207 00:08:13,060 --> 00:08:15,100 It is made up of different pyrrole rings 208 00:08:15,100 --> 00:08:16,610 that are bridged together. 209 00:08:16,610 --> 00:08:18,730 And, if you look at this shape, there's 210 00:08:18,730 --> 00:08:21,670 a nice hole in the center, which is perfect for inserting 211 00:08:21,670 --> 00:08:23,350 a metal center. 212 00:08:23,350 --> 00:08:25,350 And pyrroles are found-- or porphyrins are found 213 00:08:25,350 --> 00:08:27,400 in many different contexts. 214 00:08:27,400 --> 00:08:28,650 So you can find them in hemes. 215 00:08:28,650 --> 00:08:30,424 Obviously, we've said that already. 216 00:08:30,424 --> 00:08:32,549 So, if you put an iron center in the middle of that 217 00:08:32,549 --> 00:08:35,400 and you can add different functional groups 218 00:08:35,400 --> 00:08:38,309 to the outside of the pyrrole, then you can make a heme. 219 00:08:38,309 --> 00:08:41,159 In chlorophyll, it has a magnesium ion 220 00:08:41,159 --> 00:08:42,450 in the center of it. 221 00:08:42,450 --> 00:08:45,510 Enzymes have these to hold a whole bunch 222 00:08:45,510 --> 00:08:48,900 of different metal centers or even to not hold any metal 223 00:08:48,900 --> 00:08:55,610 centers, just the very common organic group. 224 00:08:55,610 --> 00:08:58,880 The organic chemists said, wow, these enzymes 225 00:08:58,880 --> 00:09:00,883 use all of these porphyrin groups, 226 00:09:00,883 --> 00:09:03,050 and it does some really interesting redox chemistry. 227 00:09:03,050 --> 00:09:04,830 So we can make these in the lab. 228 00:09:04,830 --> 00:09:07,790 So they have made synthetic versions of porphyrins 229 00:09:07,790 --> 00:09:10,670 with manganese, iron, and cobalt that 230 00:09:10,670 --> 00:09:12,680 catalyze a bunch of reactions in the lab, 231 00:09:12,680 --> 00:09:14,940 not necessarily in nature. 232 00:09:14,940 --> 00:09:17,480 And they're also found in petroleum complexed 233 00:09:17,480 --> 00:09:18,665 with vanadium and nickel. 234 00:09:18,665 --> 00:09:21,290 And one of the ways that they're used in the petroleum industry 235 00:09:21,290 --> 00:09:23,330 is to fingerprint petroleum. 236 00:09:23,330 --> 00:09:27,230 So, by analyzing the composition of the porphyrin 237 00:09:27,230 --> 00:09:30,020 complexes in the petroleum, they can detect 238 00:09:30,020 --> 00:09:31,160 the source of oil spills. 239 00:09:31,160 --> 00:09:33,890 They use it in geochemistry and forensics and things like that. 240 00:09:37,050 --> 00:09:39,470 So now, if we look back at our heme molecule, 241 00:09:39,470 --> 00:09:42,050 you can see that all of the hemes 242 00:09:42,050 --> 00:09:44,800 contain this porphyrin structure. 243 00:09:44,800 --> 00:09:48,490 And then you can add different groups around the outside, 244 00:09:48,490 --> 00:09:50,950 and that will change the structure. 245 00:09:50,950 --> 00:09:53,550 And that will change the function 246 00:09:53,550 --> 00:09:55,300 and change the way that it is able to bind 247 00:09:55,300 --> 00:09:56,217 to different proteins. 248 00:09:56,217 --> 00:09:59,140 So some of the hemes are just kind of stuck and held together 249 00:09:59,140 --> 00:10:01,140 with non-covalent interactions. 250 00:10:01,140 --> 00:10:04,000 And some hemes can actually be covalently bound to proteins, 251 00:10:04,000 --> 00:10:06,353 depending on how they are functionalized. 252 00:10:06,353 --> 00:10:08,770 And, like we said, they're good for oxygen binding, oxygen 253 00:10:08,770 --> 00:10:13,013 transport, and oxygen activation. 254 00:10:13,013 --> 00:10:14,930 And one of the interesting things that I found 255 00:10:14,930 --> 00:10:18,543 was the role of hemes in Impossible meat. 256 00:10:18,543 --> 00:10:20,210 So have you guys heard of the Impossible 257 00:10:20,210 --> 00:10:22,502 meat or the Impossible Burgers, the Impossible Whopper? 258 00:10:22,502 --> 00:10:24,200 Has anybody tried it? 259 00:10:24,200 --> 00:10:24,950 Yeah? 260 00:10:24,950 --> 00:10:27,092 What are-- is it actually like meat? 261 00:10:27,092 --> 00:10:27,800 What do we think? 262 00:10:27,800 --> 00:10:28,733 Is it good? 263 00:10:28,733 --> 00:10:29,900 I haven't had it personally. 264 00:10:29,900 --> 00:10:30,560 AUDIENCE: It's pretty good. 265 00:10:30,560 --> 00:10:32,080 SARAH HEWETT: Pretty good. 266 00:10:32,080 --> 00:10:32,740 Mixed reviews? 267 00:10:32,740 --> 00:10:34,712 Not so great? 268 00:10:34,712 --> 00:10:36,670 I haven't actually gotten the chance to try it, 269 00:10:36,670 --> 00:10:40,120 but an interesting thing, chemically speaking, 270 00:10:40,120 --> 00:10:42,940 about the Impossible meat is that, if you're 271 00:10:42,940 --> 00:10:45,490 going to have normal hamburger from a cow, 272 00:10:45,490 --> 00:10:48,760 then one of the proteins that is highly abundant in red meat 273 00:10:48,760 --> 00:10:49,985 is myoglobin. 274 00:10:49,985 --> 00:10:51,610 And this is the structure of myoglobin. 275 00:10:51,610 --> 00:10:54,010 And you can see that this group here 276 00:10:54,010 --> 00:10:59,050 that is not part of the peptide arrangement is a heme. 277 00:10:59,050 --> 00:11:00,520 And that happens to be what gives 278 00:11:00,520 --> 00:11:02,000 red meat a lot of its flavor. 279 00:11:02,000 --> 00:11:03,820 So, when you cook it, it releases the heme, 280 00:11:03,820 --> 00:11:08,400 and the iron center provides a lot of flavor. 281 00:11:08,400 --> 00:11:12,080 So what they did was they found that, in soybean roots, 282 00:11:12,080 --> 00:11:15,830 they have a protein called leghemoglobin, which 283 00:11:15,830 --> 00:11:18,530 is very similar in structure to myoglobin 284 00:11:18,530 --> 00:11:21,420 and also contains a heme center. 285 00:11:21,420 --> 00:11:25,320 And so, if they extract this protein out of the soybean 286 00:11:25,320 --> 00:11:28,590 plant roots, then they can add it to the Impossible meat. 287 00:11:28,590 --> 00:11:30,870 And then you will have a plant-based source of heme 288 00:11:30,870 --> 00:11:33,660 that will give you the meaty, irony flavor that we 289 00:11:33,660 --> 00:11:35,820 have associated with red meat. 290 00:11:35,820 --> 00:11:39,150 So they take proteins from soybeans and potatoes, 291 00:11:39,150 --> 00:11:41,820 like they make most vegetarian protein options out 292 00:11:41,820 --> 00:11:45,520 of, and then add this leghemoglobin from the soybean 293 00:11:45,520 --> 00:11:46,020 roots. 294 00:11:46,020 --> 00:11:47,370 And it is a red color. 295 00:11:47,370 --> 00:11:48,450 So it's also a colorant. 296 00:11:50,958 --> 00:11:53,250 The heme center is what gives your blood its red color. 297 00:11:53,250 --> 00:11:58,440 So having those conjugated pi systems in the porphyrin 298 00:11:58,440 --> 00:12:02,160 molecule and combined with an iron-- or a metal center 299 00:12:02,160 --> 00:12:03,660 will give you colors. 300 00:12:03,660 --> 00:12:06,360 And so it gives it the color and the flavor of actual meat. 301 00:12:06,360 --> 00:12:10,950 So that's an interesting role of hemes outside 302 00:12:10,950 --> 00:12:13,780 of your blood or catalase. 303 00:12:13,780 --> 00:12:17,135 Going back to catalase, again, this is the overall structure. 304 00:12:17,135 --> 00:12:19,010 And it is very hard to see with these colors, 305 00:12:19,010 --> 00:12:21,470 but there are heme centers-- you can see them-- 306 00:12:21,470 --> 00:12:25,640 in each of the four subunits of catalase. 307 00:12:25,640 --> 00:12:28,910 And it contains also a binding site for NADPH. 308 00:12:28,910 --> 00:12:32,390 And this is the structure of NADPH. 309 00:12:32,390 --> 00:12:38,660 And the NADPH's role is to maintain the oxidation 310 00:12:38,660 --> 00:12:39,920 state of the iron center. 311 00:12:39,920 --> 00:12:42,680 So it is pretty important that the iron center maintains 312 00:12:42,680 --> 00:12:44,450 its iron 3 oxidation state. 313 00:12:44,450 --> 00:12:47,417 It goes between iron 3 and iron 4 while it is active, 314 00:12:47,417 --> 00:12:49,250 but, in order for it to start the chemistry, 315 00:12:49,250 --> 00:12:50,167 it needs to be iron 3. 316 00:12:50,167 --> 00:12:52,310 And so the NADPH can donate electrons 317 00:12:52,310 --> 00:12:55,490 to make sure that the iron is in its active form. 318 00:12:57,972 --> 00:12:59,680 Now, if we want to take a moment and look 319 00:12:59,680 --> 00:13:04,570 at the actual mechanism for the reaction of hydrogen peroxide 320 00:13:04,570 --> 00:13:10,060 with catalase, this is a very pared down version 321 00:13:10,060 --> 00:13:12,080 of the catalase activation site. 322 00:13:12,080 --> 00:13:14,943 So there is a histidine residue here. 323 00:13:14,943 --> 00:13:16,610 You have your iron center and your heme. 324 00:13:16,610 --> 00:13:19,270 And then, when hydrogen peroxide comes in, 325 00:13:19,270 --> 00:13:22,750 the first step is that those electrons 326 00:13:22,750 --> 00:13:27,430 can remove one of the hydrogens from the hydrogen peroxide. 327 00:13:27,430 --> 00:13:31,740 And then this oxygen will bind to your metal center. 328 00:13:31,740 --> 00:13:34,310 So then you have your histidine now that is protonated. 329 00:13:34,310 --> 00:13:38,660 You have your oxygen bound to your metal center. 330 00:13:38,660 --> 00:13:44,590 And then this oxygen can take the hydrogen. 331 00:13:44,590 --> 00:13:48,550 And the iron center can donate some electrons here 332 00:13:48,550 --> 00:13:55,930 to form water and an iron-oxygen double bond. 333 00:13:55,930 --> 00:13:58,450 And now this is iron 4. 334 00:13:58,450 --> 00:14:00,233 So iron has donated some electrons 335 00:14:00,233 --> 00:14:01,900 to this oxygen center, and it's actually 336 00:14:01,900 --> 00:14:05,308 a radical and a cation. 337 00:14:05,308 --> 00:14:06,350 So that's the first step. 338 00:14:06,350 --> 00:14:07,990 And so that's where our first hydrogen peroxide 339 00:14:07,990 --> 00:14:09,782 molecule comes in because, if you remember, 340 00:14:09,782 --> 00:14:12,070 the overall reaction that we're working with 341 00:14:12,070 --> 00:14:18,160 is two hydrogen peroxides goes to two waters and one oxygen. 342 00:14:18,160 --> 00:14:22,100 And then the second step is, once you have this iron 343 00:14:22,100 --> 00:14:25,450 radical cation, there are two proposed mechanisms 344 00:14:25,450 --> 00:14:27,130 for how this reaction can go. 345 00:14:27,130 --> 00:14:28,990 And it can either be also mediated 346 00:14:28,990 --> 00:14:31,780 by this histidine residue where one of the hydrogens 347 00:14:31,780 --> 00:14:33,320 ends up on the histidine. 348 00:14:33,320 --> 00:14:37,830 The other one adds to the oxygen bound to the metal center. 349 00:14:37,830 --> 00:14:40,680 You release the oxygen, and this hydrogen comes back in. 350 00:14:40,680 --> 00:14:42,240 And you form your water. 351 00:14:42,240 --> 00:14:45,330 Or you can kind of ignore this histidine 352 00:14:45,330 --> 00:14:49,080 and do two hydrogen transfers onto this oxygen bound 353 00:14:49,080 --> 00:14:50,550 to the iron. 354 00:14:50,550 --> 00:14:52,680 And then you make your water and your oxygen. 355 00:14:52,680 --> 00:14:56,700 And they have done isotopic labeling studies where 356 00:14:56,700 --> 00:14:59,160 you can make this first complex and then 357 00:14:59,160 --> 00:15:01,530 add in hydrogen peroxide that's been labeled 358 00:15:01,530 --> 00:15:04,200 with oxygen-18 isotopes. 359 00:15:04,200 --> 00:15:07,830 And they have found that all of the oxygen that is produced 360 00:15:07,830 --> 00:15:09,390 is oxygen-18. 361 00:15:09,390 --> 00:15:13,110 So they know that the oxygen here comes from this peroxide. 362 00:15:13,110 --> 00:15:15,990 And then the oxygen that is bound to the iron 363 00:15:15,990 --> 00:15:17,467 ends up in the water. 364 00:15:17,467 --> 00:15:19,800 So that's kind of how they were able to narrow down some 365 00:15:19,800 --> 00:15:22,870 of the mechanisms for this. 366 00:15:22,870 --> 00:15:27,060 And, if you remember, our reaction, 367 00:15:27,060 --> 00:15:30,820 our decomposition of hydrogen peroxide can happen on its own. 368 00:15:30,820 --> 00:15:35,965 So here is a mechanism for how it may happen without catalase. 369 00:15:35,965 --> 00:15:37,920 So you can compare. 370 00:15:37,920 --> 00:15:39,360 One of the proposed mechanisms is 371 00:15:39,360 --> 00:15:40,980 that the hydrogen peroxide breaks 372 00:15:40,980 --> 00:15:42,880 into two hydroxy radicals. 373 00:15:42,880 --> 00:15:47,222 Then the second molecule of hydrogen peroxide 374 00:15:47,222 --> 00:15:48,930 reacts with one of those hydroxy radicals 375 00:15:48,930 --> 00:15:53,590 to form one molecule of water and this peroxy radical. 376 00:15:53,590 --> 00:15:56,500 And then you have one of these and one 377 00:15:56,500 --> 00:15:57,560 of these that are formed. 378 00:15:57,560 --> 00:16:00,100 So those can react and form your water and then 379 00:16:00,100 --> 00:16:03,790 your molecular oxygen. So it takes a few more steps. 380 00:16:03,790 --> 00:16:06,650 It's a little bit slower, but that 381 00:16:06,650 --> 00:16:09,720 is a comparison of how the enzyme can catalyze 382 00:16:09,720 --> 00:16:11,850 this reaction to happen much faster 383 00:16:11,850 --> 00:16:13,940 than what may happen in nature. 384 00:16:17,980 --> 00:16:21,460 So that is what is happening in the decomposition of hydrogen 385 00:16:21,460 --> 00:16:24,490 peroxide, either just hanging out on the bench top 386 00:16:24,490 --> 00:16:28,540 or with your catalase catalyzation. 387 00:16:28,540 --> 00:16:31,360 And now we're going to talk about our goals for day three 388 00:16:31,360 --> 00:16:33,470 and four of catalase. 389 00:16:33,470 --> 00:16:35,030 So, after day one and day two, you 390 00:16:35,030 --> 00:16:36,450 have seen this reaction happen. 391 00:16:36,450 --> 00:16:38,180 And now, for days three and four, 392 00:16:38,180 --> 00:16:41,150 we are going to try to quantify the amount of catalase 393 00:16:41,150 --> 00:16:42,992 in an unknown protein sample. 394 00:16:42,992 --> 00:16:45,200 And then we were going to quantify the amount of iron 395 00:16:45,200 --> 00:16:46,940 that is present in our sample of catalase 396 00:16:46,940 --> 00:16:49,148 and try to figure out how many iron centers there are 397 00:16:49,148 --> 00:16:52,010 per one molecule of catalase. 398 00:16:52,010 --> 00:16:54,960 And, based on the work of a lot of other scientists, 399 00:16:54,960 --> 00:16:57,110 we know that, theoretically, it should be how many? 400 00:17:03,660 --> 00:17:04,589 We can go back. 401 00:17:08,290 --> 00:17:11,470 So there are how many subunits in catalase? 402 00:17:11,470 --> 00:17:11,980 Four. 403 00:17:11,980 --> 00:17:14,310 How many hemes per subunit? 404 00:17:14,310 --> 00:17:14,810 One. 405 00:17:14,810 --> 00:17:18,025 So how many total iron centers in a catalase protein? 406 00:17:18,025 --> 00:17:18,650 AUDIENCE: Four. 407 00:17:18,650 --> 00:17:20,688 SARAH HEWETT: Four, excellent. 408 00:17:20,688 --> 00:17:22,230 So we know that there should be four. 409 00:17:22,230 --> 00:17:24,430 And hopefully-- that is our theoretical value. 410 00:17:24,430 --> 00:17:27,060 Hopefully, you guys are going to be able to get that 411 00:17:27,060 --> 00:17:29,170 as your answer in the lab. 412 00:17:29,170 --> 00:17:31,140 So how are we going to do that? 413 00:17:31,140 --> 00:17:33,990 First, we're going to talk about how to quantify proteins. 414 00:17:33,990 --> 00:17:37,620 So it's important to be able to determine the amount of protein 415 00:17:37,620 --> 00:17:39,480 in a given sample. 416 00:17:39,480 --> 00:17:43,480 In terms of for nutritional studies, biochemical studies, 417 00:17:43,480 --> 00:17:46,305 you need to know how much protein that you have. 418 00:17:46,305 --> 00:17:47,930 And there are a couple of ways that you 419 00:17:47,930 --> 00:17:49,930 can determine the amount of protein in a sample. 420 00:17:49,930 --> 00:17:52,340 You can do a nonspecific assay, which 421 00:17:52,340 --> 00:17:55,350 will just tell you how much overall protein that you have. 422 00:17:55,350 --> 00:17:58,760 It's not specific to certain proteins. 423 00:17:58,760 --> 00:18:01,850 And some examples of those are the biuret assay. 424 00:18:01,850 --> 00:18:03,510 You can use UV spectroscopy. 425 00:18:03,510 --> 00:18:07,850 So we did visible spectroscopy with our phosphate samples 426 00:18:07,850 --> 00:18:09,560 in the Charles River lab. 427 00:18:09,560 --> 00:18:11,390 If you use ultraviolet light, you 428 00:18:11,390 --> 00:18:14,740 can detect the amount of protein that you have. 429 00:18:14,740 --> 00:18:20,880 So some of the amino acids have the aromatic residues or the pi 430 00:18:20,880 --> 00:18:21,380 bonds. 431 00:18:21,380 --> 00:18:23,730 And those will absorb UV light. 432 00:18:23,730 --> 00:18:26,030 So you can quantify proteins that way. 433 00:18:26,030 --> 00:18:28,130 Or you can do a Bradford assay, which 434 00:18:28,130 --> 00:18:31,400 is more similar to the assay that we 435 00:18:31,400 --> 00:18:34,760 did with our phosphate samples in that it is a visible color 436 00:18:34,760 --> 00:18:37,390 change that you can see and same with the biuret assay. 437 00:18:37,390 --> 00:18:39,140 And the Bradford assay is actually the one 438 00:18:39,140 --> 00:18:40,610 that we're going to do in lab. 439 00:18:40,610 --> 00:18:42,420 So we'll talk about that more in a second. 440 00:18:42,420 --> 00:18:44,930 And, if you want to know specifically what-- 441 00:18:44,930 --> 00:18:47,553 if you want to identify the amount of one specific protein, 442 00:18:47,553 --> 00:18:49,970 you can do protein-specific assays such as a Western blood 443 00:18:49,970 --> 00:18:51,350 or an ELISA. 444 00:18:51,350 --> 00:18:55,190 And those use antibodies to select for specific proteins. 445 00:18:55,190 --> 00:18:57,800 And then you can either attach a fluorescent tag 446 00:18:57,800 --> 00:19:00,320 to the protein or a color-changing tag. 447 00:19:00,320 --> 00:19:04,130 So, when the antibody matches up with your protein, 448 00:19:04,130 --> 00:19:07,370 you get a color change or some light that you can quantify. 449 00:19:07,370 --> 00:19:11,090 And you can also do protein mass spectrometry and look at-- 450 00:19:11,090 --> 00:19:14,240 you can use that to quantify the specific proteins also. 451 00:19:17,320 --> 00:19:19,043 So the Bradford assay is the assay 452 00:19:19,043 --> 00:19:20,710 that we're going to be doing in the lab. 453 00:19:20,710 --> 00:19:23,890 And the Bradford assay uses Coomassie dye. 454 00:19:23,890 --> 00:19:28,840 And this is the structure of Coomassie dye. 455 00:19:28,840 --> 00:19:31,390 And, if you just pour the Coomassie dye out 456 00:19:31,390 --> 00:19:35,880 of the bottle, it is this brownish sort of reddish color. 457 00:19:35,880 --> 00:19:40,860 And, when it reacts to proteins, it turns blue. 458 00:19:40,860 --> 00:19:44,040 So you can quantify the amount of blue. 459 00:19:44,040 --> 00:19:45,930 And that will tell you how much protein 460 00:19:45,930 --> 00:19:47,430 you have in your sample. 461 00:19:47,430 --> 00:19:51,660 And the protein reacts with these sulfonyl groups over here 462 00:19:51,660 --> 00:19:53,140 and causes the color change. 463 00:19:53,140 --> 00:19:56,310 So, when you have all of these aromatic systems 464 00:19:56,310 --> 00:19:58,650 and electron-rich groups when you 465 00:19:58,650 --> 00:20:01,570 change the electronic structure by bonding to a protein, 466 00:20:01,570 --> 00:20:04,710 then you can change the color. 467 00:20:04,710 --> 00:20:07,490 So this is what we are going to do. 468 00:20:07,490 --> 00:20:11,230 And, in order to quantify the amount of blue or amount 469 00:20:11,230 --> 00:20:14,830 of protein that we have, similar to our phosphate analysis 470 00:20:14,830 --> 00:20:16,900 before, we need to make a series of standards. 471 00:20:16,900 --> 00:20:20,260 So you need to know what your absorbance is 472 00:20:20,260 --> 00:20:22,290 at different concentrations of the thing 473 00:20:22,290 --> 00:20:23,540 that you're trying to measure. 474 00:20:23,540 --> 00:20:24,998 So we made our phosphate standards, 475 00:20:24,998 --> 00:20:27,280 and those also happened to turn blue. 476 00:20:27,280 --> 00:20:29,530 And we are going to make a series of protein standards 477 00:20:29,530 --> 00:20:32,350 so that we know what color our Coomassie dye will 478 00:20:32,350 --> 00:20:35,170 turn a different protein concentrations. 479 00:20:35,170 --> 00:20:39,190 To do this, we are going to use bovine serum albumin. 480 00:20:39,190 --> 00:20:40,690 And it's a serum albumin protein, 481 00:20:40,690 --> 00:20:42,370 which is found in blood. 482 00:20:42,370 --> 00:20:44,980 And bovine means that the one that we're going to be using 483 00:20:44,980 --> 00:20:48,070 is coming from cows. 484 00:20:48,070 --> 00:20:51,700 And that's helpful because there is a lot of cow blood left over 485 00:20:51,700 --> 00:20:53,410 from the meat industry. 486 00:20:53,410 --> 00:20:55,060 And it is found in the blood plasma, 487 00:20:55,060 --> 00:20:57,440 and it maintains-- its role in a living animal 488 00:20:57,440 --> 00:20:59,440 is to maintain the osmotic pressure in the blood 489 00:20:59,440 --> 00:21:02,200 and carry biologically important molecules through your blood 490 00:21:02,200 --> 00:21:04,570 plasma, but we are going to use it 491 00:21:04,570 --> 00:21:08,200 as a primary standard for our protein quantification. 492 00:21:08,200 --> 00:21:11,680 And we can do this because it is abundant, inexpensive, stable, 493 00:21:11,680 --> 00:21:13,180 and reacts well with Coomassie dye. 494 00:21:13,180 --> 00:21:15,070 So we can get a nice calibration curve. 495 00:21:15,070 --> 00:21:17,800 And it's also similar enough to our protein, 496 00:21:17,800 --> 00:21:22,260 catalase, that we can use it to make our standard curve. 497 00:21:22,260 --> 00:21:26,482 So typically you would try to use the same molecule 498 00:21:26,482 --> 00:21:28,440 that you are quantifying to make your standard, 499 00:21:28,440 --> 00:21:30,550 but that's not always possible. 500 00:21:30,550 --> 00:21:33,600 So, in that case, it's helpful to have 501 00:21:33,600 --> 00:21:36,660 a protein that can kind of stand in, is abundant, 502 00:21:36,660 --> 00:21:37,675 and easily quantifiable. 503 00:21:37,675 --> 00:21:39,300 So you can buy different concentrations 504 00:21:39,300 --> 00:21:42,480 of this Bovine Serum Albumin, or BSA, 505 00:21:42,480 --> 00:21:44,703 from a bunch of different chemical suppliers. 506 00:21:44,703 --> 00:21:46,620 So you know what the concentration is, and you 507 00:21:46,620 --> 00:21:50,130 can use that as your standard. 508 00:21:50,130 --> 00:21:52,590 The way that we are going to do this in the lab 509 00:21:52,590 --> 00:21:55,710 is to prepare standards very similarly to how 510 00:21:55,710 --> 00:21:58,500 you did for your phosphate. 511 00:21:58,500 --> 00:22:01,260 You will have your BSA stock solution. 512 00:22:01,260 --> 00:22:04,980 And the BSA that we're going to be using is in solution form. 513 00:22:04,980 --> 00:22:09,420 And the stock solution is 2,000 micrograms per milliliter. 514 00:22:09,420 --> 00:22:12,642 And you will be diluting it with buffer. 515 00:22:12,642 --> 00:22:14,850 So again it's really important to use buffer any time 516 00:22:14,850 --> 00:22:16,225 that you're working with proteins 517 00:22:16,225 --> 00:22:18,810 because of the different structures 518 00:22:18,810 --> 00:22:21,500 that we showed you before. 519 00:22:21,500 --> 00:22:24,630 The side chains could be charged. 520 00:22:24,630 --> 00:22:27,710 And that will impact their interactions with each other. 521 00:22:27,710 --> 00:22:29,810 So you want to make sure that the pH is correct 522 00:22:29,810 --> 00:22:32,227 so that your protonation states of all of your amino acids 523 00:22:32,227 --> 00:22:35,450 are what they should be in terms of like a physiological pH. 524 00:22:35,450 --> 00:22:38,900 So you'll add-- dilute your BSA with buffer. 525 00:22:38,900 --> 00:22:42,170 And then you will have a bunch of different standards 526 00:22:42,170 --> 00:22:43,370 at varying concentrations. 527 00:22:43,370 --> 00:22:45,628 And then you'll have one where you don't add any BSA, 528 00:22:45,628 --> 00:22:46,670 and it's just the buffer. 529 00:22:46,670 --> 00:22:48,305 And that will serve as your blank. 530 00:22:52,515 --> 00:22:55,140 The way this is going to work in lab is that you will pipette-- 531 00:22:55,140 --> 00:22:56,880 you'll be using little microcentrifuge 532 00:22:56,880 --> 00:22:58,800 tubes and the micropipetters. 533 00:22:58,800 --> 00:23:02,010 And so this is a very quantitative experiment. 534 00:23:02,010 --> 00:23:04,775 So you need to get really good with your micropipetting 535 00:23:04,775 --> 00:23:07,400 technique and make sure that you don't contaminate your samples 536 00:23:07,400 --> 00:23:08,817 because you're going to be working 537 00:23:08,817 --> 00:23:11,430 with very small quantities, microliters of things. 538 00:23:11,430 --> 00:23:13,350 So you want to make sure that you 539 00:23:13,350 --> 00:23:15,840 are changing your pipette tips at an appropriate time 540 00:23:15,840 --> 00:23:20,440 and using the pipettes accurately. 541 00:23:20,440 --> 00:23:22,997 So you will pipette the standards 542 00:23:22,997 --> 00:23:24,330 into your microcentrifuge tubes. 543 00:23:24,330 --> 00:23:27,390 And then we will give you a standard sample-- 544 00:23:27,390 --> 00:23:29,280 or an unknown sample of catalase. 545 00:23:29,280 --> 00:23:32,490 And then you will put catalase in each 546 00:23:32,490 --> 00:23:33,900 of the microcentrifuge tubes. 547 00:23:33,900 --> 00:23:35,983 And we're going to do five samples of the catalase 548 00:23:35,983 --> 00:23:37,710 so that we have five unknowns that you 549 00:23:37,710 --> 00:23:42,650 can do an analysis of your error on that, 550 00:23:42,650 --> 00:23:45,310 similar to the phosphate where we ran five standards from-- 551 00:23:45,310 --> 00:23:46,290 or five samples from the river. 552 00:23:46,290 --> 00:23:48,060 You guys saw how much those can fluctuate 553 00:23:48,060 --> 00:23:50,160 depending on how you prepared the samples 554 00:23:50,160 --> 00:23:52,380 or how well you pipetted. 555 00:23:52,380 --> 00:23:54,330 So it'll be the same thing. 556 00:23:54,330 --> 00:24:01,470 We will have multiples, multiple trials. 557 00:24:01,470 --> 00:24:03,120 Then you will add the Coomassie reagent 558 00:24:03,120 --> 00:24:05,860 to each of the standards and your samples. 559 00:24:05,860 --> 00:24:08,350 And this is where it is going to be very important that you 560 00:24:08,350 --> 00:24:11,242 pay attention to the timing of this experiment. 561 00:24:11,242 --> 00:24:12,700 So you will add your Coomassie dye. 562 00:24:12,700 --> 00:24:14,492 And then you will shake it up a little bit. 563 00:24:14,492 --> 00:24:19,460 And then, as soon as you add the dye, you will start a timer. 564 00:24:19,460 --> 00:24:22,265 And you will close the tubes, shake them, allow the color 565 00:24:22,265 --> 00:24:23,390 to develop for two minutes. 566 00:24:23,390 --> 00:24:26,810 And then, at two minutes, you will pour all of your samples 567 00:24:26,810 --> 00:24:29,360 into cuvettes. 568 00:24:29,360 --> 00:24:32,720 And, in this case, we will be using the smaller cuvettes 569 00:24:32,720 --> 00:24:35,540 than what you guys used for the last samples, 570 00:24:35,540 --> 00:24:38,210 but they go in the same instrument. 571 00:24:38,210 --> 00:24:40,700 You will transfer your samples to the cuvettes. 572 00:24:40,700 --> 00:24:44,720 And then you will measure the absorbance at 595 nanometers. 573 00:24:44,720 --> 00:24:46,580 And the program on the UV/Vis instruments 574 00:24:46,580 --> 00:24:49,410 are set up to go to that wavelength 575 00:24:49,410 --> 00:24:51,240 when you click on it. 576 00:24:51,240 --> 00:24:53,490 You need to start running your samples at 10 minutes. 577 00:24:53,490 --> 00:24:54,660 So this is why it's really important. 578 00:24:54,660 --> 00:24:56,493 So, once you start adding the Coomassie dye, 579 00:24:56,493 --> 00:24:59,115 you need to start a timer because the dye is-- 580 00:24:59,115 --> 00:25:00,740 it's important that all of your samples 581 00:25:00,740 --> 00:25:05,317 incubate for the same amount of time so that it's consistent. 582 00:25:05,317 --> 00:25:07,400 So you don't want to take your time like adding it 583 00:25:07,400 --> 00:25:09,733 to the beginning samples so that, by the time you add it 584 00:25:09,733 --> 00:25:12,680 to your unknowns, it's been like five minutes. 585 00:25:12,680 --> 00:25:15,660 So you want to work quickly and carefully. 586 00:25:15,660 --> 00:25:18,190 The dye is most sensitive around 10 minutes after addition. 587 00:25:18,190 --> 00:25:20,190 So that's when you will get really good spectra. 588 00:25:20,190 --> 00:25:21,930 If you wait too long, then you will 589 00:25:21,930 --> 00:25:26,160 see that the protein-dye complex will start to precipitate out 590 00:25:26,160 --> 00:25:27,030 of your solution. 591 00:25:27,030 --> 00:25:31,578 And you'll get these black chunks in your cuvettes. 592 00:25:31,578 --> 00:25:33,870 And that's when you know that you can no longer measure 593 00:25:33,870 --> 00:25:37,590 the absorbance of your sample. 594 00:25:37,590 --> 00:25:40,043 So that'll be careful, and we will 595 00:25:40,043 --> 00:25:41,960 try to stagger people so that not everybody is 596 00:25:41,960 --> 00:25:45,562 using the UV/Vis at the same time so that you have your-- 597 00:25:45,562 --> 00:25:47,770 you can do everything in the allotted amount of time. 598 00:25:50,563 --> 00:25:51,980 You will make a calibration curve, 599 00:25:51,980 --> 00:25:53,910 and it will look something like this. 600 00:25:53,910 --> 00:25:57,380 And, as you can see, it's not the most linear thing. 601 00:25:57,380 --> 00:26:01,190 BSA has three linear regions when 602 00:26:01,190 --> 00:26:03,230 you are doing a Bradford assay. 603 00:26:03,230 --> 00:26:07,228 And the first one is from 0 to 125 micrograms per milliliter. 604 00:26:07,228 --> 00:26:09,020 We don't really have any data points there, 605 00:26:09,020 --> 00:26:11,480 and your protein concentration in your catalase sample 606 00:26:11,480 --> 00:26:12,605 should be higher than that. 607 00:26:12,605 --> 00:26:14,820 So we're not really going to look at that region. 608 00:26:14,820 --> 00:26:17,990 And then the other region is in the middle here, 609 00:26:17,990 --> 00:26:23,750 either from 125 to 1,000 or 125 to 750 micrograms 610 00:26:23,750 --> 00:26:25,820 per milliliter. 611 00:26:25,820 --> 00:26:29,720 And, when you get your data, you can look at this and graph 612 00:26:29,720 --> 00:26:32,060 both of these regions to see which 613 00:26:32,060 --> 00:26:34,700 one gives you a more linear and more steeper slope. 614 00:26:34,700 --> 00:26:38,510 And that's the one that you can use to calculate 615 00:26:38,510 --> 00:26:40,020 your unknown samples with. 616 00:26:40,020 --> 00:26:42,650 And the third region is above 1,000 micrograms 617 00:26:42,650 --> 00:26:44,150 per milliliter, but, as you can see, 618 00:26:44,150 --> 00:26:47,550 you're getting above one absorbance unit there. 619 00:26:47,550 --> 00:26:50,295 And so then it's not as accurate beyond there. 620 00:26:50,295 --> 00:26:51,920 So we're not going to worry about that. 621 00:26:54,093 --> 00:26:55,510 So, when you get your data, you'll 622 00:26:55,510 --> 00:26:58,057 need to figure out where it is most linear, where 623 00:26:58,057 --> 00:26:59,140 the slope is the steepest. 624 00:26:59,140 --> 00:27:00,890 And that is what you will use to calculate 625 00:27:00,890 --> 00:27:02,994 your unknown concentrations. 626 00:27:06,710 --> 00:27:09,400 And then it is just like you did for phosphate, 627 00:27:09,400 --> 00:27:11,900 and you'll be able to calculate your unknown concentrations. 628 00:27:11,900 --> 00:27:14,480 Any questions about the quantification 629 00:27:14,480 --> 00:27:19,330 of the amount of catalase protein? 630 00:27:22,360 --> 00:27:24,730 So our calibration curve, also one thing to note, 631 00:27:24,730 --> 00:27:26,740 is in units of micrograms per milliliter. 632 00:27:26,740 --> 00:27:28,750 And you'll know how many milliliters of solution 633 00:27:28,750 --> 00:27:29,470 that you added. 634 00:27:29,470 --> 00:27:31,810 And we know that the molecular weight of catalase 635 00:27:31,810 --> 00:27:36,500 is 240 kilodaltons or 240,000 grams per mole. 636 00:27:36,500 --> 00:27:39,790 So you can figure out how many moles of catalase protein 637 00:27:39,790 --> 00:27:43,570 you have from the concentration and that conversion factor. 638 00:27:43,570 --> 00:27:46,150 So that's something to note. 639 00:27:46,150 --> 00:27:50,110 And then, once we have our amount of catalase protein, 640 00:27:50,110 --> 00:27:53,740 we can determine the amount of iron in the protein. 641 00:27:53,740 --> 00:27:57,740 So this is-- and we'll be doing that using the ferrozine assay. 642 00:27:57,740 --> 00:28:00,910 This is the structure of ferrozine. 643 00:28:00,910 --> 00:28:07,990 And the ferrozine molecule by itself is colorless. 644 00:28:07,990 --> 00:28:10,150 And then, when you complex it with iron, 645 00:28:10,150 --> 00:28:12,680 it turns into a magenta color. 646 00:28:12,680 --> 00:28:16,774 And there are three molecules of ferrozine for every one iron 647 00:28:16,774 --> 00:28:18,106 ion. 648 00:28:18,106 --> 00:28:22,220 And so it forms in an octahedral sort of geometry. 649 00:28:22,220 --> 00:28:24,070 So, if there are six binding sites on iron, 650 00:28:24,070 --> 00:28:31,250 then you'll have one ferrozine here, one there, and one there. 651 00:28:31,250 --> 00:28:35,630 And it bonds to these two nitrogen centers here. 652 00:28:35,630 --> 00:28:37,460 And again, like the Coomassie dye, 653 00:28:37,460 --> 00:28:41,030 there are a lot of pi electrons and aromatic systems, which 654 00:28:41,030 --> 00:28:42,490 help to give it its color. 655 00:28:45,195 --> 00:28:46,570 So the way that we're going to do 656 00:28:46,570 --> 00:28:50,888 this is we will be using again UV/Vis spectroscopy 657 00:28:50,888 --> 00:28:53,430 because we have something that goes from colorless to colored 658 00:28:53,430 --> 00:28:55,590 when it interacts with our molecule of interest 659 00:28:55,590 --> 00:28:57,250 or our ion of interest. 660 00:28:57,250 --> 00:28:58,868 So the first step in this procedure 661 00:28:58,868 --> 00:29:00,660 is going to be adding methanesulfonic acid. 662 00:29:00,660 --> 00:29:03,780 And then you will heat it at 104 degrees for 40 minutes. 663 00:29:03,780 --> 00:29:09,100 And it's very important that you keep the temperature range 664 00:29:09,100 --> 00:29:10,425 around 104 degrees. 665 00:29:10,425 --> 00:29:11,800 And you don't want to go too high 666 00:29:11,800 --> 00:29:15,160 because we'll be heating them in the microcentrifuge tubes. 667 00:29:15,160 --> 00:29:17,650 And, if you heat it too hot, then your centrifuge tube 668 00:29:17,650 --> 00:29:19,480 can boil over, or it can build up pressure, 669 00:29:19,480 --> 00:29:20,522 and the cap will pop off. 670 00:29:20,522 --> 00:29:23,050 And you'll lose all of your solution. 671 00:29:23,050 --> 00:29:24,550 So you need to be careful with that. 672 00:29:24,550 --> 00:29:27,880 And the purpose of this step is to release the iron 673 00:29:27,880 --> 00:29:28,955 from the heme group. 674 00:29:28,955 --> 00:29:31,330 So, when you add a bunch of acid, the iron gets released. 675 00:29:31,330 --> 00:29:32,913 And then we can quantify it because it 676 00:29:32,913 --> 00:29:35,910 won't be bound to our heme. 677 00:29:35,910 --> 00:29:38,160 Then we will add sodium hydroxide 678 00:29:38,160 --> 00:29:40,880 to neutralize the acid. 679 00:29:40,880 --> 00:29:44,450 And then you're going to get a ferrozine complex mixture that 680 00:29:44,450 --> 00:29:46,468 will be prepared by your TAs. 681 00:29:46,468 --> 00:29:48,260 And you will add it to all of your samples. 682 00:29:48,260 --> 00:29:51,200 And the mixture is ascorbic acid, 683 00:29:51,200 --> 00:29:56,360 which reduces the iron 3 to iron 2+ because that is what will 684 00:29:56,360 --> 00:29:59,300 complex with our ferrozine molecule. 685 00:29:59,300 --> 00:30:02,810 Ammonium acetate helps to buffer the pH. 686 00:30:02,810 --> 00:30:06,120 Neocuproine binds to any copper ions that are in the solution. 687 00:30:06,120 --> 00:30:11,992 So the ferrozine ligand is not necessarily specific to iron. 688 00:30:11,992 --> 00:30:13,700 So, if there are any copper contaminants, 689 00:30:13,700 --> 00:30:16,010 then it will also bind to copper, 690 00:30:16,010 --> 00:30:19,410 but the neocuproine is more specific for copper. 691 00:30:19,410 --> 00:30:21,800 So it will bind to the copper ions 692 00:30:21,800 --> 00:30:24,752 and make sure that it does not contaminate your assay. 693 00:30:24,752 --> 00:30:26,210 And then, of course, the ferrozine, 694 00:30:26,210 --> 00:30:28,293 that binds to the iron to make the colored complex 695 00:30:28,293 --> 00:30:29,510 that we are going to measure. 696 00:30:32,190 --> 00:30:36,632 And then the-- so the measurement of the ferrozine 697 00:30:36,632 --> 00:30:38,090 is going to be pretty much the same 698 00:30:38,090 --> 00:30:39,357 as the BSA and the phosphate. 699 00:30:39,357 --> 00:30:40,940 Similar to UV/Vis, you'll make a bunch 700 00:30:40,940 --> 00:30:43,670 of standards of iron sulfate. 701 00:30:43,670 --> 00:30:47,140 And then you will have your catalase samples 702 00:30:47,140 --> 00:30:48,340 that have iron in them. 703 00:30:48,340 --> 00:30:50,610 And then you will make your standard curve. 704 00:30:50,610 --> 00:30:52,120 The ferrozine is linear. 705 00:30:52,120 --> 00:30:54,670 So you'll be able to make your standard curve, just graph it, 706 00:30:54,670 --> 00:30:56,553 and use the line. 707 00:30:56,553 --> 00:30:58,970 And then you will be able to use the equation of your line 708 00:30:58,970 --> 00:31:02,930 to determine your concentration of your unknown samples 709 00:31:02,930 --> 00:31:05,110 from your catalase. 710 00:31:05,110 --> 00:31:08,710 The data analysis for this lab is 711 00:31:08,710 --> 00:31:10,810 going to be using the protein assay results 712 00:31:10,810 --> 00:31:11,960 and the iron assay results. 713 00:31:11,960 --> 00:31:16,240 You will determine the number of iron centers per catalase. 714 00:31:16,240 --> 00:31:19,270 And so you have the micrograms per milliliter 715 00:31:19,270 --> 00:31:21,470 of your catalase protein. 716 00:31:21,470 --> 00:31:24,370 So you can convert that using your molecular weight 717 00:31:24,370 --> 00:31:26,620 to moles of catalase. 718 00:31:26,620 --> 00:31:28,720 And then you will also get your iron 719 00:31:28,720 --> 00:31:30,430 in micrograms per milliliter. 720 00:31:30,430 --> 00:31:32,440 And, using the molecular weight of iron, 721 00:31:32,440 --> 00:31:34,060 you can convert that to moles and then 722 00:31:34,060 --> 00:31:36,550 do a mole-to-mole ratio of iron to catalase. 723 00:31:36,550 --> 00:31:39,660 And hopefully your answer is somewhere around four 724 00:31:39,660 --> 00:31:41,800 if all goes well. 725 00:31:41,800 --> 00:31:43,300 So, in your discussion, you can talk 726 00:31:43,300 --> 00:31:45,010 about sources of error, how close you 727 00:31:45,010 --> 00:31:47,710 were to the theoretical value of four, 728 00:31:47,710 --> 00:31:49,780 any discrepancies between your calculated ratio, 729 00:31:49,780 --> 00:31:50,805 the expected ratio. 730 00:31:50,805 --> 00:31:52,930 And then you'll do an error analysis of the protein 731 00:31:52,930 --> 00:31:54,050 and iron concentrations. 732 00:31:54,050 --> 00:31:56,290 So you will run five unknown samples 733 00:31:56,290 --> 00:32:00,680 of catalase and five samples with the ferrozine assay 734 00:32:00,680 --> 00:32:01,180 of iron. 735 00:32:01,180 --> 00:32:04,370 So you'll have five measurements that should be the same. 736 00:32:04,370 --> 00:32:06,275 So those you can do your average, 737 00:32:06,275 --> 00:32:08,150 your standard deviation, confidence interval. 738 00:32:08,150 --> 00:32:09,310 And then, if you have any outliers, 739 00:32:09,310 --> 00:32:11,477 that's when you can use the Q test for the outliers. 740 00:32:13,522 --> 00:32:14,980 And there was a couple of questions 741 00:32:14,980 --> 00:32:18,100 that came up during the Charles River lab report. 742 00:32:18,100 --> 00:32:19,900 When we were making the standard curves 743 00:32:19,900 --> 00:32:21,970 for the phosphate determination, people 744 00:32:21,970 --> 00:32:24,430 had some outliers in their standard curves. 745 00:32:24,430 --> 00:32:25,847 And they were trying to figure out 746 00:32:25,847 --> 00:32:29,040 how they could get rid of those or if they should. 747 00:32:29,040 --> 00:32:31,880 And the Q test only works if you're measuring the same thing 748 00:32:31,880 --> 00:32:32,693 multiple times. 749 00:32:32,693 --> 00:32:34,610 So that does not work for getting outliers out 750 00:32:34,610 --> 00:32:37,980 of a standard linear curve. 751 00:32:37,980 --> 00:32:40,640 But if you have-- in your standard curve, 752 00:32:40,640 --> 00:32:42,860 if you have one sample that is supposed 753 00:32:42,860 --> 00:32:44,630 to be a higher concentration than the one 754 00:32:44,630 --> 00:32:47,930 before it and it has a lower absorbance or something wildly 755 00:32:47,930 --> 00:32:51,383 higher, then you can look at it and say I know for a fact 756 00:32:51,383 --> 00:32:53,300 that, if something has a higher concentration, 757 00:32:53,300 --> 00:32:54,920 it should have a higher absorbance. 758 00:32:54,920 --> 00:32:56,690 I can get rid of this point. 759 00:32:56,690 --> 00:33:00,290 You can also just kind of by looking at it-- 760 00:33:00,290 --> 00:33:01,760 that's not as scientific, but you 761 00:33:01,760 --> 00:33:05,540 can make an argument for why you would remove a data point based 762 00:33:05,540 --> 00:33:10,460 on how it looks compared to the line of best fit. 763 00:33:10,460 --> 00:33:14,180 And you can also compare the R squared values of the line 764 00:33:14,180 --> 00:33:15,930 with the point and without the data point. 765 00:33:15,930 --> 00:33:17,888 And, if it gets closer to 1 when you remove it, 766 00:33:17,888 --> 00:33:19,370 then it is more linear. 767 00:33:19,370 --> 00:33:22,010 You obviously don't want to be taking out data points left 768 00:33:22,010 --> 00:33:24,093 and right until you only have like your three best 769 00:33:24,093 --> 00:33:25,910 points that give you a super straight line. 770 00:33:25,910 --> 00:33:29,423 But, if there are any that are very obviously not on the line, 771 00:33:29,423 --> 00:33:31,340 then that is one way that you can remove them. 772 00:33:35,120 --> 00:33:39,700 So yeah, are there any questions about catalase 773 00:33:39,700 --> 00:33:42,580 or any of the other labs? 774 00:33:42,580 --> 00:33:43,292 Anything? 775 00:33:43,292 --> 00:33:44,500 This is all I have for today. 776 00:33:44,500 --> 00:33:45,708 This was kind of a short one. 777 00:33:50,725 --> 00:33:51,225 No. 778 00:33:54,250 --> 00:33:58,590 All right, then those of you who are going to lab, 779 00:33:58,590 --> 00:33:59,570 you can head up there. 780 00:33:59,570 --> 00:34:02,700 And don't forget to turn your reports in. 781 00:34:02,700 --> 00:34:05,300 And yeah, the clipboard is around.