Detection of amino acids and proteins

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• Detection of amino acids and proteins
• Ultraviolet absorbance _at_ 280nm
• Recognizes Phe, Tyr, Trp, and disulfide bonds
• To quantitate- need molar absorbance coefficient
  of the protein
• Luckily a fully unfolded protein has same
  properties of absorbance close to its
  constituents in the aromatic range. So if you
  know the number of aromatics and disulfides you
  can calculate this coefficient.

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2. Staining Commonly use Coomassie Blue- a dye
which doesnt react with protein but rather
sticks to it forming a non-covalent complex. It
makes the non-covalent complex using a mix of
non-polar and ionic interactions however, this
is not true for all proteins. No one really knows
how it works.
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Two types G250- used to quantify the amount
of protein in solution because complex formation
changes their absorbance properties. In acidic
conditions the absorbance max moves from 465 to
595 nm. Very simple and widely used. The
R-group on the picture before is a methyl
group. R250- used to stain proteins in gels.
This procedure relies on the proteins being made
insoluble and binding the dye tightly. Most of
the excess dye is removed washing the gel
(methanol, acetic acid water)-- de-stain.
Approximately 0.1mg of protein can be detected on
a polyacrylamide gel. The R-group on the picture
before is a Hydrogen. For more sensitive
detection- use silver staining (10-9 g) but in
general for any type of accurate structure
determination we need more than this (x-ray and
NMR).
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• Determination of protein sizes
• A peptide becomes a protein when it has more than
  50 residues.
• So- whats the molecular weight of the protein?
  If you know the amino acid sequence then you
  simply add it up.
• Sedimentation analysis Analytical
  Ultracentrifugation
• Runs _at_ 250,000rpm
• Under a given set of conditions the rate of
  sedimentation of a protein depends on its MW,
  density, shape, and interactions with the
  solvent. A protein that is large is more likely
  to migrate rapidly but it might be aggregating.
  A small protein may move slower or it may have an
  unusual shape.
• Measure the time it takes for large molecules
  to move to bottom to determine the size.

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2. Gel Filtration Size of a protein determines
its rate of passage through a molecular
sieve. Molecular Sieve- small particles with a
network of pores into which molecules of less
than some maximum size can penetrate. The
smaller the protein-the greater the probability
it will enter the internal volume of the
particles. Pour proteins into a molecular sieve.
Then wash with a buffer. The first proteins to
elute- TOO LARGE TO ENTER PORES. The volume of
buffer required to elute them- Void Volume (Vo)
volume of column outside the particles. Other
proteins are eluted in DECREASING order of the
molecular size. A gel filtration column is
calibrated using proteins of known size. Note
Funny shapes can mess you up!
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3. SDS Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Extremely popular method. Check
protein movement in polyacrylamide gel
electrophoresis in the presence of the detergent
sodium dodecyl sulfate(SDS) This method is
related to gel filtrations in that the size of a
protein is estimated by its migration through the
small pores of a gel matrix. In this case the
gel matrix is continuous rather than particulate,
so smaller proteins move through faster. Use a
set of standard molecular weight markers to
compare your protein migration to those.
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The eletrophoretic mobility (how fast something
moves in relation to charge) of a protein is
determined by its weight, charge and
shape. Charge and shape are standardized for
all proteins by SDS electrophoresis SDS binds
to proteins (somehow), disrupts their structure
and shape, dissociates them into polypeptide
chains and imposes comparable shapes and net
charge densities on them. Dont really know how-
It just works!! Protein-SDS complexes have
electrophoretic mobilities thru polyacrylamide
gels that are inversely proportional to the
logarithm of the length of the polypeptide
chain. Compare to standards Get
your weight!
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Determining the primary amino acid sequence The
1o sequence identifies a protein unambiguously,
determines all its chemical and biological
properties and specifies its structure. 1. Edman
degradation- sequencing from the N-terminus It
removes and identifies one residue at a time.
Requires that the terminal a-amino group be free
so it can be reacted with phenylisothiocyantate
Must be in alkaline conditions
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2. Mass Spectrometry Can be used for MW
determination and with Edman degradation to help
solve the primary sequence 3. Enzyme digest Use
chymotrypsin to cleave after every Tyr, Phe,
Trp, and Leu Use tryspin to cleave after ever
Lys, Arg Once the sequence is cleaved using one
of various enzymes or procedures, use Mass Spec
on the fragments. A database is used to tell you
what each fragment is. However, most
sequencing done from gene sequences.
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Assembly of 1o structure Coded information for
the structures of proteins is contained in the
genetic material of the chromosome, usually DNA,
in the form of linear sequences of nucleotide
bases. 3 sequential nucleotides contain the code
for a single amino acid residue The genetic info
of the DNA is constantly checked and edited by
the cell to ensure that it is altered as little
as possible-only the correct amino acid is placed
in the correct place.
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Gene Structure- brief background All the info
for synthesizing the 1o structure of a protein is
encoded in the genetic material of the
chromosomes which is double stranded DNA. Info
is encoded in sequences of 4 nucleotides on one
strand A-Adenine C-Cytosine G-
Guanine T-Thymine For RNA T ? U (Uracil) The
sequence of the other strand or DNA is chemically
complementary to the first A pairs with T G
pairs with C AGCCT TCGGA Form
genes Code for proteins
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Expression of genetic info in biosynthesis of
protein 1.Chromosomal DNA undergoes
transcription to a complementary messenger RNA
(mRNA) molecule. A bunch of regulatory proteins
are present which bind to the upstream part of
the gene (promoter) or all over (enhancer
regions). Binding of the regulatory proteins to
the DNA can have positive or negative effects on
transcription? they are there to ensure that the
gene is expressed only under appropriate
conditions. When all is fine and dandy for the
expression of a gene, it is transcribed by RNA
polymerase enzymes which copies one strand of DNA
into its complementary RNA strand.
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2. Translation Mature mRNA molecules are
translated into polypeptide chains in the
cytoplasm by a complex apparatus of ribosomes,
tRNA, and various factors. Translation of mRNA
is initiated by the formation of complexes of
mRNA, the two subunits or ribosomes and 3
initiation factor proteins. Ribosomes are big-
consisting of at least 3 RNA molecules and 55
different proteins.
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Prokaryotic cells have one RNA polymerase that
transcribes all genes but its made specific for
different genes by various sigma-factor proteins.
Eukaryotic cells have 3 enzymes- RNA polymerase
I, II, and III which transcribes different
genes. I and III? genes that code for stable
RNA II? proteins Goal is to get mRNA 1.
Eukaryotic gene splicing of useless
portions-introns 2. Prokaryotic gene no
splicing MATURE mRNA
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Amino acids couple to appropriate t-RNA molecules
which adapt them and supply them in the proper
sequence to the ribosomemRNA complex where they
are attached to the polypeptide being
synthesized. SO 53 nucleotides3 dictate
what amino acid is added. This is
very important feature because we can change one
amino acid in a primary sequence to determine
which residues are the most important! Car
analogy