Biochem Lab Exam

At what voltage and for how long are we running our samples in the gel?

120V for 20-40 min

What is the name of the enzyme involved in this laboratory experiment?

Cellobiase or beta-glucosidase

Name two disease caused by misfolded proteins aggregating in the brain

Mad cow disease (BSE) Alzheimer's

What is the term used when proteins are enriched in Pro, Glu, Ser, and Thr residues? What does this indicate?

PEST factor indicates that these proteins degrade faster than other proteins

On average, how long are the half-lives of proteins that begin with Phe, Leu, Asp, Lys, or Arg? What about those that begin with Met, Ser, Ala, Thr, Val, or Gly residues?

PLALA- 3 min or less MSATVG- greater than 20 hours

In addition to loading our samples and controls in the gel, what else is important to load in the gel for reference? What information does it give you?

PAGEmark protein marker it has a mix of 12 prestained proteins of different molecular weight this helps to identify the size of the test sample proteins

How does SDS interact with proteins and what does this interaction achieve in this experiment?

SDS interacts with the amino acid in proteins causes protein to be soluble and it reverts back to its primary structure SDS coats the protein giving it a negative charge which allows it to go through the gel

How will we know if a protein is folded properly or denature in this experiment?

correctly folded proteins will emit light under UV radiation denatured proteins will not emit light under UV radiation

Why is an enzyme's shape important to its function?

enzyme's shape must be right so that the substrate can fit. Its primary structure determines its secondary or tertiary structure so if the amino acids aren't in the right orientation then the active site cannot carry out the reaction

What is the natural product of this enzyme?

glucose

What do you have to do first in order to extract intracellular material from plants?

grind the plant in a grinding tube with a pestle

What are we measuring in plants to determine their biomass?

proteins

What is the main difference between a protease enzyme and a proteasome in how they destroy proteins? Where can we find a proteasome in a eukaryotic cell?

proteins pass through the center of the proteasome where they are degraded protease enzymes cleave peptide bonds by hydrolysis Proteasomes are found in the cytosol and nucleus of a cell

Where are we getting our protein from?

provided by dr tino

Describe two roles for protein degradation

removing damaged proteins or abnormal proteins as a regulatory snap controlling the expression levels of a protein

What equipment are we using to measure this and at what wavelength?

spectrophotometer 595 nm

How does an enzyme speed up chemical reactions?

they act as a catalyst which positions the substrate by adjusting bands to become unstable and reactive which lowers the energy of activation causing the reaction to not need as much energy as it would without the enzyme

What is biodiversity?

variability among living organisms on earth, including variability between and within species and ecosystems

What is biomass?

weight of a living material expressed as dry weight per unit area

How will you be able to determine the amount of product that is produced at each time period?

when the substrate is broken down, the product will turn yellow in the basic solution the yellow can be measured by the spectrophotometer

What is the name of polymer we are using to make our protein separating gel?

polyacrylamide

When a protein is misfolded or denatured, what level of protein structure do you think it will adopt?

primary

What are the four levels of protein structure?

primary secondary tertiary quaternary

What is one practical, industrial application of this enzyme?

production of ethanol for fuel

What type of molecule is an enzyme?

protein molecules (which are made up of amino acids)

What is the small protein tag attached to "goner" proteins in the above pathway? On what amino acid residue is this attached? What would happen if you removed all the lysines from a protein through mutagenesis?

Ubiquitin e-amino group proteins wouldn't be properly degraded

What is the common degradative pathway for proteins? What are the names of the three enzymes involved, excluding the proteasome?

Ubiquitin-Proteasome Pathway E1- Ubiquitin Activating Enzyme E2- Ubiquitin Conjugating Enzyme E3- Ubiquitin Protein Ligase

What is the name of the rule that needs to be considered first when determining the lifespan of a protein? Why is it termed like that?

N-end rule AA residue on the N-terminus of a protein can be used to predict lifespan

How can you measure the rate of product formation?

calculated by determining initial slope of the graph plotted with the product produced as a function of time take the difference between two y-values and divide by time interval to give velocity in nmoles/min

What is the natural substrate of this enzyme?

cellobiose (a dissacharide)

How do chaperones assist in protein folding?

chaperones bind to the nascent polypeptide chain to prevent misfolding, by association of hydrophobic AA, chaperone then leaves and the polypeptide chain is folded into correct structure Sometimes chaperonin is required, the nascent protein is bound inside the chaperonin and in presence of ATP folds correctly and then is ejected from chaperonin

Describe the structure of a proteasome in mammalian cell. Where are proteins degraded in a proteasome?

large multimeric protein 2 copies of 14 different proteins arranged into 4 ring-like structures degraded through the center of proteasome