This preview shows page 1. Sign up to view the full content.
Unformatted text preview: CHEM 350: Introduction to Biological Chemistry
Brian Lee, Ph.D.
Ofﬁce: Neckers 146G or 324
Hours: 9:30am to 10:30am or by appointment
Textbook (required, U.S. edition only)
Fundamentals of Biochemistry, 3rd Ed., Voet, Voet & Pratt.
Study Guide (recommended)
Student Companion to Fundamentals of Biochemistry, 3rd Ed.
Tuesday 6:30 to 7:30 pm in Neckers 218
Thursday 5:00 to 6:00 pm in Neckers 410 Announcements
Undergraduate Research Opportunities
Research for credit (such as CHEM 396 or CHEM 496)
Student worker ($8.00 per hour) (http://www.siu.edu/~fao/jobs/)
Undergraduate Assistantships (http://www.siu.edu/~fao/jobs/)
McNair Scholars Program (http://www.siu.edu/~mcnair)
REACH Awards Competition (http://www.siu.edu/~reach/)
Summer Research Experiences for Undergraduates (REU)
For other REU programs, search the National Science Foundation site:
Students must contact the individual sites for information and
application materials. NSF does not have application materials and does
not select student participants. A contact person and contact
information is listed for each site. Assignments
Read Chapter 5 Proteins
Chapter 5 Problems
• First Midterm Exam, Monday February 6th
– Chapters 1 through 5 • Help Desk
Tuesday 6:30-7:30pm in Neckers 218
Thursday 5:00-6:00pm in Neckers 410 Column chromatography:
takes advantage of the size,
net charge and binding
properties of proteins.
Proteins migrate depending
on their properties.
HPLC – high performance
uses increase pressure
and small bead size to
FPLC – fast protein liquid
ﬂow rate with low pressure
pumps. Ion-exchange chromatography
(cation or anion exchange) Cation exchange:
Positive charged solutes
bind to the column.
Negative charged solutes
pas through without
interacting with the column. Elution of positive charged solute with salt gradient. Figure 5-6 Size-exclusion chromatography
(gel ﬁltration) separates proteins
according to size Small molecules “explore” the porous beads, slowing elution. Figure 5-7 Afﬁnity chromatography
separates proteins based
on their binding
separation by charge and size
PAGE – polyacrylamide gel electrophoresis
SDS – a negatively charged detergent VZ
μ = electrophoretic mobility
V = molecule velocity
E = electric potential
Z = net charge
f = frictional coefﬁcient Electrophoresis
- Stain (Coomassie brilliant blue, silver)
- autoradiograph (35S radioisotope)
- immuno-blotting (Western blot) Denaturing conditions to separate by size only
SDS-PAGE (sodium dodecyl sulfate)
Reducing agent (2-mercaptoethanol) Isoelectric Focusing using a pH gradient in the gel
Native conditions – proteins are folded
Protein migration stops when pH = pI 2D gel separation by
- isoelectric point
- molecular weight Amino acid sequence of bovine insulin.
The sequence was determined by
Frederick Sanger who developed the
ﬁrst methods to sequence proteins.
Sequencing insulin took ten years.
Protein sequencing is faster, but still
limited to short peptides (<100 residues) Protein sequencing strategy
-separate chains -digest polypeptide
-reconstruct from overlap
-repeat without reducing
agent to ﬁnd cross-links Figure 5-12 Sanger’s Reagent – (1-Fluoro-2,4-dinitrobenzene)
DNFB – reacts with free amino groups of intact peptide
- after hydrolysis DNP-amino acid identiﬁed Box 5-1 Pehr Edman’s
Edman degradation Phenylisothiocyanate (PITC)
PITC cleaves the peptide bond.
N-terminal amino acid is labeled
and can be separated by washing.
After removal, the procedure is
repeated to identify the next residue.
Figure 5-15 DNFB
2,4-dinitroﬂuorobenzene Sanger method
relied on incomplete
hydrolysis of protein Edman
can use a
PITC = phenylisothiocyanate N-terminal
identiﬁcation Figure 5-13 Reduction of disulﬁde bonds with 2-mercaptoethanol Page 106 Free thiol groups can be protected by acetylation
with iodoacetate to prevent oxidation and cross-linking Page 107 Disulﬁde bonds
interfere with the
sequencing procedure Peptide digestion with sequence speciﬁc proteases Trypsin cleaves after a positively charged residue:
Arginine or Lysine
The next residue can be anything but Proline
Page 107 Table 5-3 Chemical cleavge by
after Methionine Protein sequencing strategy – overlapping fragments Figure 5-18 Other methods for obtaining an amino acid sequence:
1) Indirectly from the DNA sequence: 2) Directly using mass spectrometry:
tandem mass spectrometry Peptide fragments can
be separated by their
mass to charge ratio Figure 5-16b Tandem mass spectrometry:
1) separation of peptide fragments
2) collision with He produces smaller fragments
3) i dentify smaller fragments with 2nd mass spectrometer
4) fragments size matched to list of possible sequences Table 5-4 Protein sequences and evolutionary relationships: Sequence alignments show amino acid residues that are
conserved by evolution. Why are some residues conserved?
Sequences show evolutionary relationships between organisms The above sequences suggest that archae and eukaryotes are
closer relatives than eubacteria such as E. coli and B. subtilis Cytochrome c
comparison Phylogenetic tree based on cytochrome c comparison Eukaryotes Myoglobin
4 subunits Figure 5-22 Hemoglobin evolved
from gene duplication
of early globin gene
to produce subunits. Variable rates of evolution
depend on protein function
blood plasma and
extracellular matrix oxygen transport electron transport and apoptosis DNA packaging into chromosomes Figure 5-23 Shared protein domains
Most are derived from
gene duplication Figure 5-24 ...
View Full Document