Biochemistry Biochemical Techniques and Methods 2 — Questions and Answers
Question 1: In enzyme kinetics, the Michaelis constant (Km) is defined as:
- The maximum reaction velocity
- The substrate concentration at half-maximal velocity (Correct answer)
- The equilibrium constant for enzyme-substrate binding
- The rate constant for product release
Correct answer: The substrate concentration at half-maximal velocity
Km is the substrate concentration at which reaction velocity equals Vmax/2; it is a measure of substrate affinity (lower Km = higher affinity) but is not a true equilibrium dissociation constant.
Question 2: Competitive inhibition of an enzyme:
- Decreases Vmax without changing Km
- Increases apparent Km without changing Vmax (Correct answer)
- Decreases both Km and Vmax
- Increases both Km and Vmax
Correct answer: Increases apparent Km without changing Vmax
A competitive inhibitor competes with substrate for the active site, increasing apparent Km (reduced affinity) but leaving Vmax unchanged because excess substrate can overcome inhibition.
Question 3: Gel filtration (size-exclusion) chromatography separates proteins such that:
- Smaller proteins elute first
- Larger proteins elute first (Correct answer)
- Proteins elute in order of charge
- Proteins elute in order of hydrophobicity
Correct answer: Larger proteins elute first
Larger proteins cannot enter the pores of the gel matrix and pass through the column faster (elute first), while smaller proteins enter the pores and take longer to elute.
Question 4: Reverse transcriptase PCR (RT-PCR) begins with which step?
- Amplification of genomic DNA
- Conversion of mRNA to complementary DNA (cDNA) (Correct answer)
- Denaturation of double-stranded DNA
- Ligation of DNA fragments
Correct answer: Conversion of mRNA to complementary DNA (cDNA)
RT-PCR starts by using reverse transcriptase to synthesize a complementary DNA (cDNA) copy of the mRNA template, which can then be amplified by conventional PCR.
Question 5: The Bradford assay measures protein concentration using which dye?
- Biuret reagent (copper sulfate)
- Coomassie Brilliant Blue G-250 (Correct answer)
- BCA (bicinchoninic acid)
- Lowry reagent
Correct answer: Coomassie Brilliant Blue G-250
The Bradford assay uses Coomassie Brilliant Blue G-250, which shifts its absorbance maximum from 465 nm to 595 nm upon binding to proteins; the absorbance at 595 nm is proportional to protein concentration.
Question 6: Southern blotting detects:
- Specific RNA sequences
- Specific DNA sequences (Correct answer)
- Specific protein antigens
- Specific lipid molecules
Correct answer: Specific DNA sequences
Southern blotting (named after Edward Southern) involves electrophoresis of restriction-digested DNA, transfer to a membrane, and hybridization with a labeled DNA probe to detect specific sequences.
Biochemistry Biochemical Techniques and Methods 2
In enzyme kinetics, the Michaelis constant (Km) is defined as: