Tymo_c40_620-637hr 2-12-2008 10:12 Page 620

SECTION

17
Experimental
Biochemistry
Learning Objectives
✓ How are proteins purified?
✓ How is the primary structure of a protein determined?
✓ What immunological techniques are used in biochemistry laboratories?
✓ How can a protein-encoding gene be cloned?
✓ How can a DNA molecule be sequenced and amplified?

Y ou have learned much about how cells, tissues, and organisms function in
your study of biochemistry. What you have learned has been presented as
facts in a textbook. But everything that is now believed to be true was at some
point simply an experimental observation and often a controversial one at that.
For instance, recall the rejection letter that Hans Krebs received when he first sub-
mitted a paper describing the citric acid cycle (p. 290). This section describes some
of the techniques used by researchers to tease information out of the cell.
Although scientists are most interested in how biochemistry take place in an
organism, this goal is technically very difficult to achieve. How can we learn about
a particular biomolecule—a protein, for instance—when it is surrounded by thou-
sands of other molecules and interacting with them? To circumvent this difficulty,
Tymo_c40_620-637hr 2-12-2008 10:12 Page 621

Ser Tyr Gln Val lle Cys Arg Asp Glu Lys Thr Gln Met lle Tyr Gln Gln His Gln Ser
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Ala Gln Cys His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Thr Cys Gln Gln Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu Gly Phe Ala
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

Gly Lys Cys Cys Glu lle Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gln Gly lle Ser Tyr
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

Arg Gly Asp Trp Ser Thr Ala Glu Ser Gly Ala Glu Cys Thr Asp Trp Gln Ser Ser Ala
101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Leu Ala Gln Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala lle Arg Leu Gly Leu Gly Asn
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140

His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp Cys
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

at least initially, the first approach to understanding how any biomolecule works Tyr
181
Phe
182
Gly
183
Asn
184
Gly
185
Ser
186
Ala
187
Tyr
188
Arg
189
Gly
190
Thr
191
His
192
Ser
193
Leu
194
Thr
195
Glu
196
Ser
197
Gly
198
Ala
199
Ser
200

Cys Leu Pro Trp Asn Ser Met lle Leu lle Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220

is to isolate and purify the biomolecule and examine its biochemically properties Cys
221
Gln
222
Ala
223
Leu
224
Gly
225
Leu
226
Gly
227
Lys
228
His
229
Asn
230
Tyr
231
Cys
232
Arg
233
Asn
234
Pro
235
Asp
236
Gly
237
Asp
238
Ala
239
Lys
240

Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser

in vitro. Toward this end, we will first examine how proteins, the workhorses of bio- 241

Cys
261
242

Ser
262
243

Thr
263
244

Cys
264
245

Gly
265
246

Leu
266
247

Arg
267
248

Gln
268
249

Tyr
269
250

Ser
270
251

Gln
271
252

Pro
272
253

Gln
273
254

Phe
274
255

Arg
275
256

lle
276
257

Lys
277
258

Gly
278
259

Gly
279
260

Leu
280

chemistry, are purified. Protein purification is both a science and an art. Phe
281

Pro
Ala
282

Gly
Asp
283

Glu
lle
284

Arg
Ala
285

Phe
Ser
286

Leu
His
287

Cys
Pro
288

Gly
Trp
289

Gly
Gln
290

lle
Ala
291

Leu
Ala
292

lle
Leu
293

Ser
Phe
294

Ser
Ala
295

Cys
Ala
296

Trp
Ala
297

lle
Ala
298

Leu
Ala
299

Ser
Ser
300

Ala
301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320

Researchers take advantage of often slight differences in the physical character- Ala
321
His
322
Cys
323
Phe
324
Gln
325
Glu
326
Arg
327
Phe
328
Pro
329
Pro
330
His
331
His
332
Leu
333
Thr
334
Val
335
Trp
336
lle
337
Leu
338
Ser
339
Ala
340

Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu Lys Tyr lle Val Lys

istics of similar proteins to separate them from one another. After a protein has 341

Lys
361
342

Glu
362
343

Phe
363
344

Asp
364
345

Asp
365
346

Asp
366
347

Thr
367
348

Tyr
368
349

Asp
369
350

Asn
370
351

Asp
371
352

lle
372
353

Ala
373
354

Leu
374
355

Leu
375
356

Gln
376
357

Leu
377
358

Lys
378
359

Ser
379
360

Asp
380

been purified, a key initial characterization is determination of its primary struc-

Ser Ser Arg Cys Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp
381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400

Leu Gln Leu Pro Asp Thp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

ture. Knowing the primary structure of a protein can be a source of insight into Ser
421
Pro
422
Phe
423
Tyr
424
Ser
425
Glu
426
Arg
427
Leu
428
Lys
429
Glu
430
Ala
431
His
432
Val
433
Arg
434
Leu
435
Tyr
436
Pro
437
Ser
438
Ser
439
Arg
440

Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp
441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

the structure and function of the protein and allows us to compare it with other Thr
461
Arg
462
Ser
463
Gly
464
Gly
465
Pro
466
Gln
467
Ala
468
Asn
469
Leu
470
His
471
Asp
472
Ala
473
Cys
474
Gln
475
Gly
476
Asp
477
Ser
478
Gly
479
Gly
480

Pro Leu Val Cys Leu Asn Asp Gly Arg Met Tyr Leu Val Gly lle lle Ser Trp Gly Leu

similar proteins. Protein purification and primary structure determination are the 481

Gly
482

Cys
483

Gly
484

Gln
485

Lys
486

Asp
487

Val
488

Pro
489

Gly
490

Val
491

Tyr
492

Thr
493

Lys
494

Val
495

Thr
496

Asn
497

Tyr
498

Leu
499

Asp
500

Trp
501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

subjects of Chapter 40. lle

521
Arg
522
Asp
523
Asn
524
Met
525
Arg
526
Pro
527

In Chapter 41, we will examine additional techniques for the investigation of Chapter 40: Techniques in
biomolecules. We will explore powerful immunological techniques that can be Protein Biochemistry
used to further investigate proteins as well as other biomolecules. These same
techniques are also useful in clinical settings for diagnosis and treatment. Finally,
we will investigate recombinant DNA technology—the tools and techniques that
allow researchers to move and connect genes and large pieces of DNA. We will
learn how genes are cloned and look into the variety of experimental and clinical
opportunities that cloning provides.

Chapter 41: Immunological and

Recombinant DNA Techniques
Tymo_c40_620-637hr 2-12-2008 10:12 Page 622

CHAPTER
40 Techniques in Protein
Biochemistry
40.1 The Proteome Is the Functional Ser
1
Tyr
2
Gln
3
Val
4
lle
5
Cys
6
Arg
7
Asp
8
Glu
9
Lys
10
Thr
11
Gln
12
Met
13
lle
14
Tyr
15
Gln
16
Gln
17
His
18
Gln
19
Ser
20

Representation of the Genome Trp

21
Leu
22
Arg
23
Pro
24
Val
25
Leu
26
Arg
27
Ser
28
Asn
29
Arg
30
Val
31
Glu
32
Tyr
33
Cys
34
Trp
35
Cys
36
Asn
37
Ser
38
Gly
39
Arg
40

40.2 The Purification of Proteins Is the Ala

41
Gln
42
Cys
43
His
44
Ser
45
Val
46
Pro
47
Val
48
Lys
49
Ser
50
Cys
51
Ser
52
Glu
53
Pro
54
Arg
55
Cys
56
Phe
57
Asn
58
Gly
59
Gly
60

First Step in Understanding Their Thr

61
Cys
62
Gln
63
Gln
64
Ala
65
Leu
66
Tyr
67
Phe
68
Ser
69
Asp
70
Phe
71
Val
72
Cys
73
Gln
74
Cys
75
Pro
76
Glu
77
Gly
78
Phe
79
Ala
80

Function Gly Lys Cys Cys Glu lle Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gln Gly lle Ser Tyr
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

40.3 Determining Primary Structure Arg

101
Gly
102
Asp
103
Trp
104
Ser
105
Thr
106
Ala
107
Glu
108
Ser
109
Gly
110
Ala
111
Glu
112
Cys
113
Thr
114
Asp
115
Trp
116
Gln
117
Ser
118
Ser
119
Ala
120

Facilitates an Understanding of Leu Ala Gln Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala lle Arg Leu Gly Leu Gly Asn
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140
Protein Function His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp Cys
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200

Cys Leu Pro Trp Asn Ser Met lle Leu lle Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220

Cys Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser
241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro Gln Phe Arg lle Lys Gly Gly Leu
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280

Phe Ala Asp lle Ala Ser His Pro Trp Gln Ala Ala Leu Phe Ala Ala Ala Ala Ala Ser
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

Pro Gly Glu Arg Phe Leu Cys Gly Gly lle Leu lle Ser Ser Cys Trp lle Leu Ser Ala
301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320

Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Trp lle Leu Ser Ala
321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340

Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu Lys Tyr lle Val Lys
341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp lle Ala Leu Leu Gln Leu Lys Ser Asp
361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380

Ser Ser Arg Cys Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp
381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400

Leu Gln Leu Pro Asp Thp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu Tyr Pro Ser Ser Arg
421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440

Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp
441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

Thr Arg Ser Gly Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly
461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

Pro Leu Val Cys Leu Asn Asp Gly Arg Met Tyr Leu Val Gly lle lle Ser Trp Gly Leu
481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500

Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu Asp Trp
501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

lle Arg Asp Asn Met Arg Pro

521 522 523 524 525 526 527

The amino acid sequence of tenecteplase, a fibrinolytic for the

acute treatment of myocardial infarction. [After X. Rabasseda,
Drugs Today 37(11):749, 2001.]

M uch of our study of biochemistry has focused on protein structure and func-
tion. We have observed that proteins are indeed the workhorses of the cell.
All of the information that we have learned about proteins raises an interesting
question: How do we know what we know about proteins? The first step toward
learning how proteins work in the cell is to learn how they work outside the cell,
in vitro. To do so, the proteins must be separated from all of the other constituents
of the cell so that their biochemical properties can be identified and characterized.
In other words, the protein must be purified.
In this chapter, we will examine some of the key techniques of protein purifi-
cation. All of these techniques take advantage of biochemical properties unique
to each protein. Then, we will learn how one crucial property of proteins—amino
acid sequence, or primary structure—is elucidated.
622
Tymo_c40_620-637hr 2-12-2008 10:12 Page 623

623
40.1 The Proteome Is the Functional Representation of the 40.2 Protein Purification
Genome
Every year, researchers are increasing their knowledge of the exact DNA base
sequences and volume of information contained in the genomes of many organ-
isms. For example, researchers recently concluded that the roundworm Caenorhab-
ditis elegans has a genome of 97 million bases and about 19,000 protein-encoding
genes, whereas that of the fruit fly Drosophilia melanogaster contains 180 million
bases and about 14,000 genes. The completely sequenced human genome contains
3 billion bases and about 25,000 genes. But this genomic knowledge is analogous
to a list of parts for a car: it does not explain which parts are present in different
components or how the parts work together. A new word, the proteome, has been
coined to signify a more complex level of information content—the level of func-
tional information, which encompasses the types, functions, and interactions of
proteins that yield a functional unit.
The term proteome is derived from proteins expressed by the genome. The
genome provides a list of gene products that could be present, but only a subset of
these gene products will actually be expressed in a given biological context. The
proteome tells us what is functionally present—for example, which proteins inter-
act to form a signal-transduction pathway or an ion channel in a membrane.
Unlike the genome, the proteome is not a fixed characteristic of the cell. Rather,
because it represents the functional expression of information, it varies with cell
type, developmental stage, and environmental conditions, such as the presence of
hormones. Almost all gene products are proteins that can be chemically modified
in a variety of ways. Furthermore, these proteins do not exist in isolation; they often
interact with one another to form complexes with specific functional properties.
An understanding of the proteome is acquired by investigating, characteriz-
ing, and cataloging proteins. In some, but not all, cases, this process begins by sep-
arating a particular protein from all other biomolecules in the cell.

40.2 The Purification of Proteins Is the First Step

in Understanding Their Function
To understand a protein—its amino acid sequence, its three-dimensional struc-
ture, and how it functions in normal and pathological states—we need to purify
the protein. In other words, we need to isolate the protein of interest from the
thousands of other proteins in the cell. This protein sample may be only a frac-
tion of 1% of the starting material, whether that starting material consists of cells
in culture or a particular organ from a plant or animal. This task is rather daunt-
ing and requires much ingenuity and patience, but, before we can even undertake
the task, we need a test that identifies the protein in which we are interested. We
will use this test after each stage of purification to see if the purification is work-
ing. Such a test is called an assay, and it is based on some unique identifying prop-
erty of the protein. For enzymes, which are protein catalysts (Chapter 5), the assay
is usually based on the reaction catalyzed by the enzyme in the cell. For instance,
the enzyme lactate dehydrogenase, an important enzyme in glucose metabolism,
carries out the following reaction:

–
O O –
C O O
Lactate
dehydrogenase C
HO C H + NAD+ + NADH + H+
C
CH3 O CH3
Lactate Pyruvate
Tymo_c40_620-637hr 2-12-2008 10:12 Page 624

624 The product, reduced nicotinamide adenine dinucleotide (NADH), in con-

40 Techniques in Protein Biochemistry
QUICK QUIZ 1 Why is an assay
required for protein purification?
trast with the other reaction components, absorbs light at 340 nm. Consequently,
we can follow the progress of the reaction by measuring the light absorbance at
340 nm in unit time—for instance, within 1 minute after the addition of the sam-
ple that contains the enzyme. Our assay for enzyme activity during the purifica-
tion of lactate dehydrogenase is thus the increase in absorbance of light at 340 nm
observed in 1 minute. Note that the assay tells us how much enzyme activity is pre-
sent, not how much enzyme protein is present.
To be certain that our purification scheme is working, we need one additional
piece of information—the amount of total protein present in the mixture being
assayed. This measurement of the total amount of protein includes the enzyme of
interest as well as all the other proteins present, but it is not a measure of enzyme
activity. After we know both how much enzyme activity is present and how much
protein is present, we can assess the progress of our purification by measuring
the specific activity, the ratio of enzyme activity to the amount of protein in the
enzyme assay at each step of our purification. The specific activity will rise as
the protein mixture used for the assay consists to a greater and greater extent of
the protein of interest. In essence, the point of the purification is to remove all
proteins except the protein in which we are interested. Quantitatively, it means
that we want to maximize specific activity.

Centrifuge
at 500 × g
for 10 minutes

Supernatant

Homogenate
forms Pellet: Nuclear
10,000 × g
20 minutes
fraction

100,000 × g Pellet: Mitochondrial

1 hour fraction

Cytosol
(soluble proteins)

Pellet: Microsomal
fraction

Figure 40.1 Differential centrifugation. Cells are disrupted in a homogenizer and the
resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of
increasing centrifugal force. The denser material will form a pellet at lower centrifugal force
than will the less-dense material. The isolated fractions can be used for further purification.
[Photographs courtesy of Dr. S. Fleischer and Dr. B. Fleischer.]
Tymo_c40_620-637hr 2-12-2008 10:12 Page 625

Proteins Must Be Removed from the Cell to Be Purified 625

Having found an assay, we must now break open the cells, releasing the cellu- 40.2 Protein Purification
lar contents, so that we can gain access to our protein. The disruption of the
cell membranes yields a homogenate, a mixture of all of the components of the
cell but no intact cells. This mixture is centrifuged at low centrifugal force,
yielding a pellet of heavy material at the bottom of the centrifuge tube and a
lighter solution above, called supernatant (Figure 40.1). The pellet and super-
natant are called fractions because we are fractionating the homogenate. The
supernatant is again centrifuged at a greater force to yield yet another pellet
and supernatant. The procedure, called differential centrifugation, yields sev-
eral fractions of decreasing density, each still containing hundreds of different
proteins, which are assayed for the activity being purified. Usually, one fraction
will have more enzyme activity than any other fraction, and it then serves as
the source of material to which more discriminating purification techniques
are applied. The fraction that is used as a source for further purification is often
called the crude extract.

Proteins Can Be Purified According to Solubility,

Protein solubility
Size, Charge, and Binding Affinity Salting out
Proteins are purified on the bases of differences in solubility, size, charge, and spe-
cific binding affinity. Usually, protein mixtures are subjected to a series of separa- Salting in
tions, each based on a different property.

Salting out. Most proteins require some salt to dissolve, a process called salt-
Salt concentration
ing in. However, most proteins precipitate out of solution at high salt concen-
trations, an effect called salting out (Figure 40.2). Salting out is due to Figure 40.2 The dependency of protein
competition between the salt ions and the protein for water to keep the protein solubility on salt concentration. The graph
in solution (water of solvation). The salt concentration at which a protein pre- shows how altering the salt concentration
cipitates differs from one protein to another. Hence, salting out can be used to affects the solubility of a hypothetical
protein. Different proteins will display
fractionate a mixture of proteins. Unfortunately, many proteins lose their activ- different curves.
ity in the presence of such high concentrations of salt. However, the salt can be
removed by the process of dialysis. The protein–salt solution is placed in a small
bag made of a semipermeable membrane, such as a cellulose membrane, with
pores (Figure 40.3). Proteins are too large to fit through the
pores of the membrane, whereas smaller molecules and ions
such as salts can escape through the pores and emerge in the
medium outside the bag (the dialysate).

Separation by size. Gel-filtration chromatography, also called Dialysis bag

molecular exclusion chromatography, separates proteins on
the basis of size. The sample is applied to the top of a column
consisting of porous beads made of an insoluble polymer such Concentrated
as dextran, agarose, or polyacrylamide (Figure 40.4). Small solution
molecules can enter these beads, but large ones cannot, and so
those larger molecules follow a shorter path to the bottom of Buffer
the column and emerge first. Molecules that are of a size to
occasionally enter a bead will flow from the column at an
intermediate position, and small molecules, which take a
longer, more circuitous path, will exit last.

Ion-exchange chromatography. Proteins can be separated on At start of dialysis At equilibrium

the basis of their net charge by ion-exchange chromatogra- Figure 40.3 Dialysis. Protein molecules (red) are retained within
phy. If a protein has a net positive charge at pH 7, it will usu- the dialysis bag, whereas small molecules (blue) diffuse into the
ally bind to a column of beads containing negatively charged surrounding medium.
Tymo_c40_620-637hr 2-12-2008 10:12 Page 626

626
40 Techniques in Protein Carbohydrate
Biochemistry polymer bead

Small molecules
enter the
aqueous spaces
within beads

Protein
sample Large
molecules
cannot enter
Molecular beads
exclusion
gel

Flow direction

Figure 40.4 Gel-filtration chromatography. A mixture of proteins in a small volume is

applied to a column filled with porous beads. Because large proteins cannot enter the
internal volume of the beads, they emerge sooner than do small ones.

carboxylate groups, whereas a negatively charged protein will not bind to the
column (Figure 40.5). A positively charged protein bound to such a column
can then be released by increasing the concentration of salt in the buffer
poured over the column. The positively charged ions of the salt compete with
positively charged groups on the protein for binding to the column. Likewise,
a protein with a net negative charge will be bound to ion-exchange beads car-
rying positive charges and can be eluted from the column with the use of a
buffer containing salt.

− − +− Affinity chromatography. Affinity chromatography is another powerful means of

+
− −
− purifying proteins. This technique takes advantage of the fact that some proteins
++ Positively charged
− − have a high affinity for specific chemical groups or specific molecules. For exam-
+ −+ − −
protein binds to
negatively charged ple, the plant protein concanavalin A, which binds to glucose, can be purified by
− − bead passing a crude extract through a column of beads containing covalently attached
− − −
− − ++ glucose residues. Concanavalin A binds to such beads, whereas most other pro-
− − teins do not. The bound concanavalin A can then be released from the column
− − − − by adding a concentrated solution of glucose. The glucose in solution displaces
− − − − the column-attached glucose residues from binding sites on concanavalin A
−
− − + (Figure 40.6).
− + − − −
− Negatively charged
− − protein flows High-pressure liquid chromatography. The ability of column techniques to separate
− through
− + − − individual proteins, called the resolving power, can be improved substantially
−
− −
through the use of a technique called high-pressure liquid chromatography
− − (HPLC), which is an enhanced version of the column techniques already dis-
− + − +
− −
cussed. The beads that make up the column material themselves are much more
Figure 40.5 Ion-exchange finely divided and, as a consequence, there are more interaction sites and thus
chromatography. This technique separates greater resolving power. Because the column is made of finer material, pressure
proteins mainly according to their net must be applied to the column to obtain adequate flow rates. The net result is high
charge. resolution as well as rapid separation (Figure 40.7).
Tymo_c40_620-637hr 2-12-2008 10:12 Page 627

0.24
Glucose-binding
protein attaches G
G 5
to glucose
residues (G) on 0.20 1
G
beads G
G

Absorbance at 220 nm
G 0.16
GG

0.12
G 23 4
G
0.08

Addition of
glucose (G)
0.04

G
G 0

G 0 5 10
G
GG Time (minutes)
G
G
Figure 40.7 High-pressure liquid
chromatography (HPLC). Gel filtration by
HPLC clearly defines the individual
GG G Figure 40.6 Affinity chromatography. proteins because of its greater resolving
Glucose-binding G
proteins are G Affinity chromatography of power. Proteins are detected by their
released on G absorbance of 220-nm light waves:
concanavalin A (shown in yellow) on a
addition of GG solid support containing covalently (1) thyroglobulin (669 kd), (2) catalase
glucose
attached glucose residues (G). (232 kd), (3) bovine serum albumin
(67 kd), (4) ovalbumin (43 kd), and
(5) ribonuclease (13.4 kd). [After K. J. Wilson
and T. D. Schlabach. In Current Protocols in
Proteins Can Be Separated by Gel Electrophoresis and Displayed Molecular Biology, vol. 2, suppl. 41, F. M.
How can we tell whether a purification scheme is effective? One way is to demon- Ausubel, R. Brent, R. E. Kingston, D. D. Moore,
strate that the specific activity rises with each purification step. Another is to visu- J. G. Seidman, J. A. Smith, and K. Struhl, Eds.
(Wiley, 1998), p. 10.14.1.]
alize the number of proteins present at each step. The technique of gel
electrophoresis makes the latter method possible.
A molecule with a net charge will move in an electric field, a phenomenon
termed electrophoresis. The distance and speed that a protein moves in elec-
trophoresis depends on the electric-field strength, the net charge on the protein,
which is a function of the pH of the electrophoretic solution, and the shape of the
protein. Electrophoretic separations are nearly always carried out in gels, such as
polyacrylamide, because the gel serves as a molecular sieve that enhances separa-
tion. Molecules that are small compared with the pores in the gel readily move
through the gel, whereas molecules much larger than the pores are almost immo-
bile. Intermediate-size molecules move through the gel with various degrees of
ease. The electrophoresis of proteins is performed in a thin, vertical slab of poly-
acrylamide. The direction of flow is from top to bottom (Figure 40.8).
Proteins can be separated largely on the basis of mass by electrophoresis in a
polyacrylamide gel in the presence of the detergent sodium dodecyl sulfate (SDS).
The negatively charged SDS denatures proteins and binds to the denatured protein
at a constant ratio of one SDS molecule for every two amino acids in the protein.
The negative charges on the many SDS molecules bound to the
protein “swamp” the normal charge on the protein and cause all
proteins to have the same charge-to-mass ratio. Thus, proteins will Na+ SO3–
differ only in their mass. Finally, a sulfhydryl agent such as mercap- O
toethanol is added to reduce disulfide bonds and completely lin- Sodium dodecyl sulfate
earize the proteins. The SDS–protein complexes are then subjected (SDS)

627
Tymo_c40_620-637hr 2-12-2008 10:12 Page 628
628 (A)
40 Techniques in Protein
Biochemistry
Figure 40.9 The staining of proteins after
electrophoresis. Proteins subjected to
electrophoresis on an SDS–polyacrylamide
gel can be visualized by staining with
Coomassie blue. The lane on the left is a
set of marker proteins of known molecular
weight. These marker proteins have been
separated on the basis of size, with the
smaller proteins moving farther into the
gel than the larger proteins. Two different
protein mixtures are in the remaining
lanes. [Wellcome Photo Library.]
(B)
−
Mixture of
macromolecules
Electrophoresis
Direction of
+ electrophoresis
Porous gel
Figure 40.8 Polyacrylamide-gel electrophoresis. (A) Gel-electrophoresis apparatus. Typically,
several samples undergo electrophoresis on one flat polyacrylamide gel. A microliter
pipette is used to place solutions of proteins in the wells of the slab. A cover is then placed
over the gel chamber and voltage is applied. The negatively charged SDS (sodium dodecyl
sulfate)–protein complexes migrate in the direction of the anode, at the bottom of the gel.
(B) The sieving action of a porous polyacrylamide gel separates proteins according to
size, with the smallest moving most rapidly.
to electrophoresis. When the electrophoresis is complete, the proteins in the gel can
be visualized by staining them with silver or a dye such as Coomassie blue, which
reveals a series of bands (Figure 40.9). Small proteins move rapidly through the gel,
whereas large proteins stay at the top, near the point of application of the mixture.
Isoelectric focusing. Proteins can also be separated electrophoretically on the basis
of their relative contents of acidic and basic residues. The isoelectric point (pI) of
a protein is the pH at which its net charge is zero. At this pH, the protein will not
migrate in an electric field. If a mixture of proteins is subjected to electrophore-
sis in a pH gradient in a gel in the absence of SDS, each protein will move until it
reaches a position in the gel at which the pH is equal to the pI of the protein. This
method of separating proteins is called isoelectric focusing. Proteins differing by
one net charge can be separated (Figure 40.10).
Two-dimensional electrophoresis. Isoelectric focusing can be combined with
SDS–PAGE (SDS–polyacrylamide gel electrophoresis) to obtain very high resolu-
tion separations. A single sample is first subjected to isoelectric focusing. This sin-
gle-lane gel is then placed horizontally on top of an SDS–polyacrylamide slab and
subjected to electrophoresis again, in a direction perpendicular to the isoelectric
focusing, to yield a two-dimensional pattern of spots. In such a gel, proteins have
been separated in the horizontal direction on the basis of isoelectric point and in
(A)
+ − ±
Low pH − − High pH
+ ± ±
(+) ± + + (−)
−
(B)
Low pH High pH
(+) (−)
Figure 40.10 The principle of isoelectric focusing. A pH gradient is established in a gel
before the sample has been loaded. (A) The sample is loaded and voltage is applied. The
proteins will migrate to their isoelectric pH, the location at which they have no net charge.
(B) The proteins form bands that can be excised and used for further experimentation.
Tymo_c40_620-637hr 2-12-2008 10:12 Page 629

(A) (B) Isoelectric focusing

Low pH
(+)
SDS–polyacrylamide slab

SDS-PAGE
Figure 40.11 Two-dimensional gel electrophoresis. (A) A protein sample is initially
fractionated in one direction by isoelectric focusing as described in Figure 40.10. The
isoelectric focusing gel is then attached to an SDS–polyacrylamide gel, and electrophoresis
is performed in the second direction, perpendicular to the original separation. Proteins with
the same pI value are now separated on the basis of mass. (B) Proteins from E. coli were
separated by two-dimensional gel electrophoresis, resolving more than a thousand different
proteins. The proteins were first separated according to their isoelectric pH in the
horizontal direction and then by their apparent mass in the vertical direction. [(B) Courtesy of
Dr. Patrick H. O’Farrell.]

the vertical direction on the basis of mass. More than a thousand different pro-
teins in the bacterium Escherichia coli can be resolved in a single experiment by
two-dimensional electrophoresis (Figure 40.11).

A Purification Scheme Can Be Quantitatively Evaluated

Some combination of purification techniques will usually yield a pure protein. To
determine the success of a protein-purification scheme, we monitor the procedure
at each step by determining specific activity and by performing an SDS-PAGE
analysis. Consider the results for the purification of a hypothetical protein, sum-
marized in Table 40.1 and Figure 40.12. At each step, the following parameters are
measured:
• Total Protein. The quantity of protein present in a fraction is obtained by deter-
mining the protein concentration of a part of each fraction and multiplying by
the fraction’s total volume.

Table 40.1 Quantification of a purification protocol for a hypothetical protein

Total protein Total activity Specific activity Yield Purification
Step (mg) (units) (units mg-1) (%) level
Homogenization 15,000 150,000 10 100 1
Salt fractionation 4,600 138,000 30 92 3
Ion-exchange 1,278 115,500 90 77 9
chromatography
Gel-filtration 68.8 75,000 1,100 50 110
chromatography
Affinity 1.75 52,500 30,000 35 3,000
chromatography

629
Tymo_c40_620-637hr 2-12-2008 10:12 Page 630

Homogenate Salt Ion-exchange Gel-filtration Affinity

630 fractionation chromatography chromatography chromatography
40 Techniques in Protein Biochemistry 1 2 3 4 5

Figure 40.12 Electrophoretic analysis of a protein purification. The purification scheme in

Table 40.1 was analyzed by SDS-PAGE. Each lane contained 50 ␮g of sample. The
effectiveness of the purification can be seen as the band for the protein of interest becomes
more prominent relative to other bands.

QUICK QUIZ 2 What physical
differences among proteins allow for
their purification?
• Total Activity. The enzyme activity for the fraction is obtained by measuring
the enzyme activity in the volume of fraction used in the assay and multiply-
ing by the fraction’s total volume.
• Specific Activity. This parameter, obtained by dividing total activity by total pro-
tein, enables us to measure the degree of purification by comparing specific
activities after each purification step. Recall that the goal of a purification
scheme is to maximize specific activity.
• Yield. This parameter is a measure of the total activity retained after each
purification step as a percentage of the activity in the crude extract. The
amount of activity in the initial extract is taken to be 100%.
• Purification level. This parameter is a measure of the increase in purity and is
obtained by dividing the specific activity, calculated after each purification step,
by the specific activity of the initial extract.
As we see in Table 40.1, several purification steps can lead to several thousand-
fold purification. Inevitably, in each purification step, some of the protein of inter-
est is lost, and so our overall yield is 35%. A good purification scheme takes into
account purification levels as well as yield.
The SDS-PAGE depicted in Figure 40.12 shows that, if we load the same
amount of protein onto each lane after each step, the number of bands decreases
in proportion to the level of purification and the amount of protein of interest
increases as a proportion of the total protein present.

40.3 Determining Primary Structure Facilitates

an Understanding of Protein Function
An important means of characterizing a pure protein is to determine its primary
structure, which can tell us much about the protein. Recall that the primary struc-
ture of a protein is the determinant of its three-dimensional structure, which ulti-
mately determines the protein’s function. Comparison of the sequence of normal
Tymo_c40_620-637hr 2-12-2008 10:12 Page 631

631
40.3 Determining Primary Structure

R
O N
R NH2

O O
O OH
O

O
OH

Fluorescamine Amine derivative

Figure 40.13 Fluorescent derivatives of amino acids. Fluorescamine reacts with the ␣-amino
group of an amino acid to form a fluorescent derivative.

proteins with those isolated from patients with pathological conditions allows an
understanding of the molecular basis of diseases.
Let us examine first how we can sequence a simple peptide, such as
Ala-Gly-Asp-Phe-Arg-Gly
The first step is to determine the amino acid composition of the peptide. The pep-
tide is hydrolyzed into its constituent amino acids by heating it in strong acid. The
individual amino acids can then be separated by ion-exchange chromatography
and visualized by treatment with fluorescamine, which reacts with the ␣-amino
group to form a highly fluorescent product (Figure 40.13).
The concentration of an amino acid in solution is proportional to the fluorescence
of the solution. The solution is then run through a column. The amount of buffer
required to remove the amino acid from the column is compared with the elution
pattern of a standard mixture of amino acids, revealing the identity of the amino
acid in the solution (Figure 40.14). The composition of our peptide is
(Ala, Arg, Asp, Gly2, Phe)
The parentheses denote that this is the amino acid composition of the peptide,
not its sequence.
The sequence of a protein can then be determined by a process called the
Edman degradation. The Edman degradation sequentially removes one residue at

ELUTION PROFILE OF PEPTIDE HYDROLYSATE Figure 40.14 Determination of amino

acid composition. Different amino acids in
Gly
a peptide hydrolysate can be separated by
ion-exchange chromatography on a
Asp Ala Phe Arg sulfonated polystyrene resin (such as
Dowex-50). Buffers (in this case, sodium
citrate) of increasing pH are used to elute
Absorbance

the amino acids from the column. The

amount of each amino acid present is
determined from the absorbance.
NH3
Met

Phe
Asp

Leu
Glu

Cys
Pro

Arg
Ser
Thr

Gly

His
Ala

Lys
Val

Tyr
lle

Aspartate, which has an acidic side chain,

is the first to emerge, whereas arginine,
ELUTION PROFILE OF STANDARD AMINO ACIDS which has a basic side chain, is the last.
The original peptide is revealed to be
pH 3.25 pH 4.25 pH 5.28 composed of one aspartate, one alanine,
0.2 M Na citrate 0.2 M Na citrate 0.35 M Na citrate
one phenylalanine, one arginine, and two
Elution volume glycine residues.
Tymo_c40_620-637hr 2-12-2008 10:12 Page 632

O
H H
EDMAN DEGRADATION H2N Asp Phe Arg Gly
N + N
C H
1 2 3 4 5 H3C H O
S
Phenyl isothiocyanate Ala Gly
Labeling
Labeling
First
1 2 3 4 5 round
O
H H H H
Release N N Asp Phe Arg Gly
N
H
1 2 3 4 5 H3C H O
S

Labeling
Release

2 3 4 5 Second
round
S
Release
H H
NH Asp Phe Arg Gly
2 3 4 5 N + H2N
H
O
O CH3

PTH−alanine Peptide shortened by one residue

Figure 40.15 The Edman degradation. The labeled amino-terminal residue (PTH–alanine in
the first round) can be released without hydrolyzing the rest of the peptide. Hence, the
amino-terminal residue of the shortened peptide (Gly-Asp-Phe-Arg-Gly) can be determined in
the second round. Three more rounds of the Edman degradation reveal the complete
sequence of the original peptide.

a time from the amino end of a peptide (Figure 40.15). Phenyl isothiocyanate
reacts with the terminal amino group of the peptide, which then cyclizes and
breaks off the peptide, yielding an intact peptide shortened by one amino acid.
The cyclic compound is a phenylthiohydantoin (PTH)–amino acid, which can be
identified by chromatographical procedures. The Edman procedure can then be
repeated sequentially to yield the amino acid sequence of the peptide.

Table 40.2 Specific cleavage of polypeptides

Reagent Cleavage site
Chemical cleavage
Cyanogen bromide Carboxyl side of methionine residues
O-Iodosobenzoate Carboxyl side of tryptophan residues
Hydroxylamine Asparagine–glycine bonds
2-Nitro-5-thiocyanobenzoate Amino side of cysteine residues
Enzymatic cleavage
Trypsin Carboxyl side of lysine and arginine residues
Clostripain Carboxyl side of arginine residues
Staphylococcal protease Carboxyl side of aspartate and glutamate residues
(glutamate only under certain conditions)
Thrombin Carboxyl side of arginine
Chymotrypsin Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine,
and methionine
Carboxypeptidase A Amino side of C-terminal amino acid (not arginine, lysine, or
proline)

632
Tymo_c40_620-637hr 2-12-2008 10:12 Page 633

Tryptic peptides Chymotryptic peptide 633

Ala Ala Trp Gly Lys Val Lys Ala Ala Trp
40.3 Determining Primary Structure
Thr Phe Val Lys

Tryptic peptide Tryptic peptide

Thr Phe Val Lys Ala Ala Trp Gly Lys
Chymotryptic overlap peptide

Figure 40.16 Overlap peptides. The peptide obtained by chymotryptic digestion overlaps
two tryptic peptides, establishing their order.

In principle, we should be able to sequence an entire protein by using

the Edman method. In practice, the peptides cannot be much longer than about
50 residues, because the reactions of the Edman method are not 100% efficient
and, eventually, the sequencing reactions are out of order. We can circumvent this
obstacle by cleaving the original protein at specific amino acids into smaller pep-
tides that can be sequenced independently. In essence, the strategy is to divide and
conquer.
Specific cleavage can be achieved by chemical or enzymatic methods.
Table 40.2 gives several ways of specifically cleaving polypeptide chains. The pep-
tides obtained by specific chemical or enzymatic cleavage are separated, and the
sequence of each purified peptide is then determined by the Edman method. At
this point, the amino acid sequences of segments of the protein are known, but
the order of these segments is not yet defined. How can we order the peptides to
obtain the primary structure of the original protein? The necessary additional
information is obtained from overlap peptides (Figure 40.16). A second cleavage
technique is used to split the polypeptide chain at different sites. Some of the pep-
tides from the second cleavage will overlap two or more peptides from the first QUICK QUIZ 3 Differentiate
cleavage, and they can be used to establish the order of the peptides. The entire between amino acid composition
amino acid sequence of the polypeptide chain is then known. and amino acid sequence.

Amino Acid Sequences Are Sources of Many Kinds of Insight

A protein’s amino acid sequence is a valuable source of insight into the protein’s
function, structure, and history.
1. The sequence of a protein of interest can be compared with all other known
sequences to ascertain whether significant similarities exist. Does this protein belong
to an established family? A search for kinship between a newly sequenced protein
and the millions of previously sequenced ones takes only a few seconds on a per-
sonal computer. If the newly isolated protein is a member of an established class
of protein, we can begin to infer information about the protein’s structure and
function. For instance, chymotrypsin and trypsin are members of the serine pro-
tease family, a clan of proteolytic enzymes that have a common catalytic mecha-
nism based on a reactive serine residue. If the sequence of the newly isolated
protein shows sequence similarity with trypsin or chymotrypsin, the result sug-
gests that it, too, may be a serine protease.
2. Comparison of sequences of the same protein in different species yields a wealth of
information about evolutionary pathways. Genealogical relations between species
can be inferred from sequence differences between their proteins. We can even esti-
mate the time at which two evolutionary lines diverged, thanks to the clocklike
nature of random mutations. For example, a comparison of serum albumins found
in primates indicates that human beings and African apes diverged 5 million years
ago, not 30 million years ago as was once thought. Sequence analyses have opened
a new perspective on the fossil record and the pathway of human evolution.
3. Amino acid sequences can be searched for the presence of internal repeats. Such
internal repeats can reveal the history of an individual protein itself. Many
Tymo_c40_620-637hr 2-12-2008 10:12
634
40 Techniques in Protein Biochemistry
N C
Figure 40.17 Repeating motifs in a
protein chain. Calmodulin, a calcium
sensor, contains four similar units (shown
in red, yellow, blue, and orange) in a single
polypeptide chain. Notice that each unit
binds a calcium ion (shown in green).
[Drawn from 1CLL.pdb.]
Figure 40.18 Sickled red blood cells
trapped in capillaries. The micrograph
shows sickled red blood cell trapped in tiny
blood vessels called capillaries. The
trapped cells impede the flow of blood
through the tissues. Consequently, the
cells are deprived of oxygen and the
tissues are damaged. [Courtesy of National
Heart, Lung and Blood Institute.]
Page 634
proteins apparently have arisen by the duplication of primordial genes. For exam-
ple, calmodulin, a ubiquitous calcium sensor in eukaryotes, contains four similar
calcium-binding modules that arose by gene duplication (Figure 40.17).
4. Many proteins contain amino acid sequences that serve as signals designating
their destinations or controlling their processing. For example, a protein destined for
export from a cell or for location in a membrane contains a signal sequence, a
stretch of about 20 hydrophobic residues near the amino terminus that directs the
protein to the appropriate membrane. Another protein may contain a stretch of
amino acids that functions as a nuclear localization signal, directing the protein to
the nucleus.
5. Sequence data allow a molecular understanding of diseases. Many diseases
are caused by mutations in DNA that result in alterations in the amino acid
sequence of a particular protein. These alterations often compromise the protein’s
function. For instance, sickle-cell anemia is caused by a change in a single amino
acid in the primary structure of the ␤ chain of hemoglobin. Approximately 70% of
the cases of cystic fibrosis are caused by the deletion of one particular amino acid
from the 1480-amino-acid-containing protein that controls chloride transport
across cell membranes. Indeed, a major goal of biochemistry is to elucidate the mol-
ecular basis of disease with the hope that this understanding will lead to effective
treatment.
Clinical Insight
Understanding Disease at the Molecular Level: Sickle-Cell Anemia
Results from a Single Amino Acid Change
Studies with mutations of hemoglobin have provided many examples showing
that alterations in the primary structure of a protein result in pathological condi-
tions. The role of hemoglobin, which is contained in red blood cells, is to bind
oxygen in the lungs and to transport and release oxygen to tissues that require oxy-
gen for combusting fuels. If the ability of hemoglobin to carry and release oxygen
is somehow compromised, anemia results. Anemia is characterized by a host of
symptoms, most commonly fatigue. A well-studied example of anemia is sickle-
cell anemia, which is most commonly found in people from sub-Saharan Africa
or their descendants (p. 113). Recall from Chapter 8 that the defining feature of
sickle-cell anemia is that the red blood cells adopt a sickle shape after the hemo-
globin has released its bound oxygen. These sickled cells clog small capillaries and
impair blood flow. The results may be painful swelling of the extremities and a
higher risk of stroke or bacterial infection (owing to poor circulation). The sick-
led red cells also do not remain in circulation as long as normal cells do, leading
to anemia.
What is the molecular defect associated with sickle-cell anemia? A single
amino acid substitution in the ␤ chain of hemoglobin is responsible—namely,
the substitution of a valine residue for a glutamate residue in position 6. The
mutated form is referred to as hemoglobin S (Hb S). In people with sickle-cell ane-
mia, both copies of the hemoglobin ␤-chain (Hb B) gene are mutated. The Hb S
substitution substantially decreases the solubility of deoxyhemoglobin, although
it does not markedly alter the properties of oxyhemoglobin. Hence, sickling takes
place in the small capillaries after the hemoglobin has released its oxygen to the
tissues. The deoxyhemoglobin molecules associate with one another and form
insoluble aggregates that deform the cell, leading to the characteristic sickle shape
(Figure 40.18). Thus, sickle-cell anemia, like the spongiform encephalopathies
discussed earlier (Chapter 4), is a pathological condition resulting from protein
aggregation. ■
Tymo_c40_620-637hr 2-12-2008 10:12 Page 635

SUMMARY 635
Answers to Quick Quizzes
40.1 The Proteome Is the Functional Representation of the Genome
The rapid progress in gene sequencing has advanced another goal of
biochemistry—the elucidation of the proteome. The proteome is the com-
plete set of proteins expressed and includes information about how they are
modified, how they function, and how they interact with other molecules.
Unlike the genome, the proteome is not static and varies with cell type,
developmental stage, and environmental conditions.
40.2 The Purification of Proteins Is the First Step in Understanding Their
Function
Proteins can be separated from one another and from other molecules on
the basis of such characteristics as solubility, size, charge, and binding affin-
ity. SDS-PAGE separates the polypeptide chains of proteins under denatur-
ing conditions largely according to mass. Proteins can also be separated
electrophoretically on the basis of net charge by isoelectric focusing in a pH
gradient.
40.3 Determining Primary Structure Facilitates an Understanding of Protein
Function
The amino acid composition of a protein can be ascertained by hydrolyz-
ing the protein into its constituent amino acids. The amino acids can be
separated by ion-exchange chromatography and quantitated by their reac-
tion with fluorescamine. Amino acid sequences can be determined by
Edman degradation, which removes one amino acid at a time from the
amino end of a peptide. Phenyl isothiocyanate reacts with the terminal
amino group to form a phenylthiohydantoin–amino acid and a peptide
shortened by one residue. Longer polypeptide chains are broken into
shorter ones for analysis by specifically cleaving them with a reagent that
breaks the peptide at specific sites. Amino acid sequences are rich in infor-
mation concerning the kinship of proteins, their evolutionary relations, and
diseases produced by mutations. Knowledge of a sequence provides valu-
able clues to conformation and function.

Key Terms
proteome (p. 623) ion-exchange chromatography isoelectric focusing (p. 628)
assay (p. 623) (p. 625) two-dimensional electrophoresis
homogenate (p. 625) affinity chromatography (p. 626) (p. 629)
salting in (p. 625) high-pressure liquid chromatography Edman degradation (p. 631)
salting out (p. 625) (HPLC) (p. 626) phenyl isothiocyanate (p. 632)
dialysis (p. 625) gel electrophoresis (p. 627) overlap peptide (p. 633)
gel-filtration chromatography (p. 625) isoelectric point (p. 628)

Answers to QUICK QUIZZES

1. An assay, which should be based on some unique bio-
chemical property of the protein that is being purified,
allows the detection of the protein of interest.
2. Differences in size, solubility, charge, and the specific
binding of certain molecules.
3. Amino acid composition is simply the amino acids that
are present in the protein. Many proteins can have the same
amino acid composition. Amino acid sequence is the
sequence of amino acids or the primary structure of the
protein. Each protein has a unique amino acid sequence.
Tymo_c40_620-637hr 2-12-2008 10:12 Page 636

636 40 Techniques in Protein Biochemistry

Problems
1. Salting out. Why do proteins precipitate at high salt
concentrations?
2. Salting in. Although many proteins precipitate at high salt
concentrations, some proteins require salt in order to dissolve
in water. Explain why some proteins require salt to dissolve.
3. Competition for water. What types of R groups would
compete with salt ions for water of solvation?
(b) Under what conditions might the statement in part a be
incorrect?
(c) Some proteins migrate anomalously in SDS-PAGE gels.
For instance, the molecular weight determined from an
SDS-PAGE gel is sometimes very different from the molec-
ular weight determined from the amino acid sequence. Sug-
gest an explanation for this discrepancy.

4. Column choice. (a) The octapeptide AVGWRVKS was 10. A question of efficiency. The Edman method of protein
digested with the enzyme trypsin. Would ion-exchange or sequencing can be used to determine the sequence of pro-
gel-filtration chromatography be most appropriate for sep- teins no longer than approximately 50 amino acids. Why is
arating the products? Explain. (b) Suppose that the peptide this length limitation the case?
had, instead, been digested with chymotrypsin. What would
be the optimal separation technique? Explain. Chapter Integration Problem
11. Quaternary structure. A protein was purified to homo-
5. Frequently used in shampoos. The detergent sodium
geneity. Determination of the mass by gel-filtration chro-
dodecyl sulfate (SDS) denatures proteins. Suggest how SDS
matography yields 60 kd. Chromatography in the presence
destroys protein structure.
of urea yields a 30-kd species. When the chromatography is
6. Making more enzyme? In the course of purifying an repeated in the presence of urea and ␤-mercaptoethanol, a
enzyme, a researcher performs a purification step that single molecular species of 15 kd results. Describe the struc-
results in an increase in the total activity to a value greater ture of the molecule.
than that present in the original crude extract. Explain how
the amount of total activity might increase. Data Interpretation Problems
7. Protein purification problem. Complete the following 12. Protein sequencing 1. Determine the sequence of hexa-
table. peptide on the basis of the following data. Note: When the
Specific sequence is not known, a comma separates the amino acids.
Total Total activity Purifi- (See Table 40.2.)
Purification protein activity (units cation Yield Amino acid composition: (2R,A,S,V,Y)
procedure (mg) (units) mg-1) level (%)
N-terminal analysis of the hexapeptide: A
Crude 20,000 4,000,000 1 100 Trypsin digestion: (R,A,V) and (R,S,Y)
extract
Carboxypeptidase digestion: no digestion
(NH4)2SO4 5,000 3,000,000
precipitation
Chymotrypsin digestion: (A,R,V,Y) and (R,S)
DEAE– 1,500 1,000,000 13. Protein sequencing 2. Determine the sequence of a pep-
cellulose tide consisting of 14 amino acids on the basis of the follow-
chromatography ing data.
Gel-filtration 500 750,000 Amino acid composition: (4S,2L,F,G,I,K,M,T,W,Y)
chromatography N-terminal analysis: S
Affinity 45 675,000 Carboxypeptidase digestion: L
chromatography Trypsin digestion: (3S,2L,F,I,M,T,W) (G,K,S,Y)
Chymotrypsin digestion: (F,I,S) (G,K,L) (L,S) (M,T)
8. Dialysis. Suppose that you precipitate a protein with (S,W) (S,Y)
1 M (NH4)2SO4, and you wish to reduce the concentration N-terminal analysis of (F,I,S) peptide: S
of the (NH4)2SO4. You take 1 ml of your sample and dialyze Cyanogen bromide treatment: (2S,F,G,I,K,L,M*,T,Y)
it in 1000 ml of buffer. At the end of dialysis, what is the con- (2S,L,W)
centration of (NH4)2SO4 in your sample? How could you M*, methionine detected as homoserine
further lower the (NH4)2SO4 concentration?
9. Charge to mass. (a) Proteins treated with a sulfhydryl
reagent such as ␤-mercaptoethanol and dissolved in sodium
dodecyl sulfate have the same charge-to-mass ratio. Explain.
Tymo_c40_620-637hr 2-12-2008 10:12 Page 637

Answers to Problems 637

Answers to Problems
1. If the salt concentration becomes too high, the salt ions 8. The sample was diluted 1000-fold. The concentration
interact with the water molecules. Eventually, there are not after dialysis is thus 0.001 M or 1 mM. You could reduce the
enough water molecules to interact with the protein, and salt concentration by dialyzing your sample, now 1 mM, in
the protein precipitates. more buffer free of (NH4)2SO4.
2. If there is lack of salt in a protein solution, the proteins 9. (a) Because one SDS molecule binds to a protein for
may interact with one another—the positive charges on every two amino acids in the proteins, in principle, all pro-
one protein with the negative charges on another or several teins will have the same charge-to-mass ratio. For instance,
others. Such an aggregate becomes too large to be solublized a protein consisting of 200 amino acids will bind 100 SDS
by water alone. If salt is added, the salt neutralizes the charges molecules, whereas a protein consisting of 400 amino acids
on the proteins, preventing protein–protein interactions. will bind 200 SDS molecules. The average mass of an amino
3. Charged and polar R groups on the surface of the acid is 110, and there is one negative charge per SDS mole-
enzyme. cule. Thus, the charge-to-mass ratio of both proteins is the
4. (a) Trypsin cleaves after arginine (R) and lysine (K), same—0.0045. (b) The statement might be incorrect if the
generating AVGWR, VK, and S. Because they differ in size, protein contains many charged amino acids. (c) The protein
these products could be separated by molecular exclusion may be modified. For instance, serine, threonine, and tyro-
chromatography. sine may have phosphoryl groups attached.
(b) Chymotrypsin, which cleaves after large aliphatic or 10. Because the cleavage does not occur every time for each
aromatic R groups, generates two peptides of equal size peptide being sequenced. Consequently, after many repeti-
(AVGW) and (RVKS). Separation based on size would not be tions (approximately 50), many different peptides are
effective. The peptide RVKS has two positive charges (R and releasing different amino acids at the same time. To illus-
K), whereas the other peptide is neutral. Therefore, the two trate this point, assume that each sequencing step is 98%
products could be separated by ion-exchange chromatography. efficient. The proportion of correct amino acids released
after 50 rounds is 0.9850, or 0.4—a hopelessly impure mix.
5. The long hydrophobic tail on the SDS molecule (p. 627)
disrupts the hydrophobic interactions in the interior of the 11. Treatment with urea disrupts noncovalent bonds. Thus,
protein. The protein unfolds, with the hydrophobic R groups the original 60-kd protein must be made of two 30-kd sub-
now interacting with the SDS rather than with one another. units. When these subunits are treated with urea and
␤-mercaptoethanol, a single 15-kd species results, suggest-
6. An inhibitor of the enzyme being purified might have
ing that disulfide bonds link the 30-kd subunits.
been present and subsequently removed by a purification
step. This removal would lead to an apparent increase in the 12. N terminal: A
total amount of enzyme present. Trypsin digestion: Cleaves at R. Only two peptides are pro-
duced. Therefore, one R must be internal and the other
7.
Specific
must be the C-terminal amino acid. Because A is N termi-
Total Total activity Purifi- nal, the sequence of one of the peptides is AVR.
Purification protein activity (units cation Yield Carboxypeptidase digestion: No digestion confirms that R
procedure (mg) (units) mg–1) level (%) is the C-terminal amino acid.
Chymotrypsin digestion: Cleaves only at Y. Combined with
Crude 20,000 4,000,000 200 1 100 the preceding information, chymotrypsin digestion tells us
extract that the sequences of the two peptides are AVRY and SR.
(NH4)2SO4 5,000 3,000,000 600 3 75 Thus the complete peptide is AVRYSR.
precipitation
DEAE– 1,500 1,000,000 667 3.3 25
13. First amino acid: S
cellulose Last amino acid: L
chromatography Cyanogen bromide cleavage: M is 10th position, C-terminal
residues are: (2S,L,W)
Size- 500 750,000 1,500 7.5 19
exclusion
N-terminal residues: (G,K,S,Y), tryptic peptide, ends in K
chromatography N-terminal sequence: SYGK
Chymotryptic peptide order: (S,Y), (G,K,L), (F,I,S), (M,T),
Affinity 45 675,000 15,000 75 17
(S,W), (S,L)
chromatography
Sequence: SYGKLSIFTMSWSL

Selected readings for this chapter can be found online at www.whfreeman.com/Tymoczko