Experimental Techniques and Information Flashcards by Kevin Bradley

What is a buffer?

A solution containing a mixture of a weak acid (HA) and its conjugate base (A-) capable of resisting changes in pH upon the addition of additional acid or base.

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What is the Henderson-Hasselbalch equation?

pH = pKa + log(base/acid)

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What is the Henderson-Hasselbalch equation used for?

Calculating the pH of a buffer

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Beyond what range of pH change can a buffer no longer work properly?

Plus or minus 1 pH

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What does a pH meter measure?

It is a potentiometer that measures the potential difference between a glass electrode and a reference electrode (the calomel electrode)

Glass bulb only permeable to H ions, which are measured.

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When titrating an amino acids, what substances are being titrated?

Amino acid

- Water in which it dissolves acts as a base

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How can the pKa of ionizable groups on amino acids be found?

By titrating the amino acid and then creating a titration curve. Water should be titrated so that the effects of water can be corrected for.

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What is the equivalence point on an amino acid titration curve?

An inflection point where added mmoles of acid are equivalent to the mmoles of acidic-amino group present in solution

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Where are the two pka of neutral amino acids dissociable groups found on a titration curve?

Halfway between 0 mmoles HCL added and mmoles of HCL added at the equivalence point. nd an equal distance to the right of the equivalence point along the x-axis.

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What are the axis of a titration curve?

x axis: mmoles of acid added

y axis: pH of solution

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Is pKa higher for the amino group or the carboxyl group of amino acids?

Amino group (about 9.8)

Carboxyl group is about 2.4

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How can you correct for the effect of water on amino acid titration? (Give formula)

mmol AA - (mmol H20)x(Total mL AA/total mmol H20)

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When correcting for the effect of water on amino acid titration, are you converting mmols of of acid, mmols of AA or pH?

mmols of acid (eg. HCL)

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Are most proteins soluble or insoluble in water?

Soluble, cells are mostly water

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What three things can change the solubility of proteins?

Changing pH
Adding salts
Organic solvents

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When proteins are precipitated out of a solution, do they retain their function?

They will usually retain their full function when the solution is diluted with water to dissolve them again

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Does protein denaturation involve cutting covalent bonds?

No, denaturation does not result in a smaller protein

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What are four ways to denature proteins?

Boiling
Urea
Organic solvents
Detergents

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Is denaturation permanent?

Sometimes

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What is the symbol of the dissociation constant? What is the negative logarithmic value of this constant?

Ka (very small number)

Negative logarithmic value is pKa

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How many pH units above or below the pKa for a group results in an equilibrium essentially completely to the right or left?

2 pH units

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What is the pKa of the carboxyl on the two acidic amino acids R group? What are these two amino acids?

Around 4

Glutamic acid
Aspartic acid

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What is the pKa for the basic R groups of the three basic amino acids? What are the three basic amino acids?

Arginine and lysine: 11

- Histidine: 6

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Every amino acid has an alpha-carboxyl group and an alpha-amino group. What are the pKas of these two groups?

Carboxyl: 2
Amino: 9

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What is the isoelectric charge for a protein?

The pH at which the net charge of a protein is 0

How can the buffering capacity of a protein be observed?

By adding pH indicators that work in acidic or basic range and then adding acid/base. If the colour change is not very strong, then the protein is buffering against that treatment (acid or base).

At what UV absorption wavelength do the nitrogenous bases of DNA and RNA strongly absorb light?

260 nm

What is the hyperchromic effect in DNA?

Disruption of the secondary structure found in dsDNA increases UV absorption of stacked hydrogen-bonded bases. Disruption can be done by increasing the temperature, leading to an A260 closer to an equivalent number of free mononucleotides.

What property of DNA allows the GC content to be determined?

Melting temperature

DNase affects what two properties of DNA?

- Viscosity | - A260

The pores of a gel exclusion matrix become smaller as ____?

Pores become smaller as the number of cross-links between polymers increases.

During column chromatography, will small molecules or big molecules be eluted first? Why?

Big molecules. Because smaller molecules are retained in the gel matrix in the internal fluid while large molecules remain in the external phase (void volume).

What is a diffusate during dialysis?

The outside fluid that small molecules are diffusing to

How do buffers of differing ionic strength effect electrophoresis outcomes?

A low ionic strength buffer allows charge molecules to travel faster, but with less resolution than a highly ionic buffer.

What are three factors which increase electrophoretic separation?

- Higher voltage - More time allowed - Shorter distance between electrodes

What is the potential gradient of electrophoresis? What are the units?

The voltage drop divided by the distance between the electrodes. The units for this are V/cm

What does salivary amylase digest (be specific)?

The α(1-4) glycosidic bonds between glucose residues in amylose (straight chain) and amylopectin (branched) of starch.

What are the two products of amylose digestion?

Glucose and maltose

What are products of amylopectin and glycogen (starch) digestion?

- Glucose - Maltose - Isomaltose - Dextrins

What is special about dextrins?

They contain both α(1 - 4) and α(1 - 6) glycosidic bonds

How does iodine detect starch?

Winding of a left-handed helix of amylose around clusters of iodine atoms forms the dark blue iodine-starch complex

What is an enzyme unit (U)?

The amount of enzyme that will catalyze the use of 1 umol of substrate (or the formation of 1 umol of product) per minute in a reaction under defined conditions.

How is the enzyme unit (U) usually expressed?

U/mL

What is the specific activity of enzymes?

THe number of enzyme units (U) per mg of total protein (U/mg) SA = U/mg protein

True or false? The more purified an enzyme preparation is, the greater its specific activity is.

True

Why does the velocity value of enzyme level off over time? (5)

- Denaturation of enzyme - Product inhibition - Substrate depletion - Inactivation of coenzyme - Increase of the reverse reaction at the product concentration builds

When is the velocity of an enzyme reaction measured?

Only at the very beginning of a reaction Enzyme studies only use initial velocity

Why does iodine form a lighter coloured solution with glycogen than it does with starch?

Because glycogen is branched, meaning it cannot form as tight iodine-glycogen complexes as it does for iodine-starch complexes

When lactic acid dehydrogenase converts pyruvate to lactate, is it oxidizing or reducing its cofactor NADH?

Oxidizing

under anaerobic conditions, which direction is LDH working in?

Reducing pyruvate to lactate

In the case of lactic acid dehydrogenase, how is Keq calculated? (give formula)

Keq = [lactate][NAD+] / [pyruvate][NADH]

At what point of enzyme activity does free energy change apply to?

The equilibrium

How can free energy change be calculated with the equilibrium constant? (give formula)

ΔG' = -RT(lnKeq)

A very large Keq value likely indicates? (in terms of spontaneity)

A negative change in free energy and therefore a spontaneous reaction

What is the dilution formula?

(V total)(C final) = (V sample)(C initial)

What is the alanine aminotransferase reaction?

Alanine + α-ketoglutarate = pyruvate + glutamate

What is the katal (kat)?

The amount of enzyme that converts one mole of substrate per second

1 cal is equal to how many: - kcal - kilojoules

- 1 kcal | - 4.2 kJ

What is the basal metabolic rate? (include units)

kcal consumed per day (kcal/day)

Why is the BMR adjusted lower for females?

Because they have more adipose tissue and therefore a slower metabolism overall.

What is ACT? How is it calculated?

Activity level | BMR/24)(hours multiplied by activity level, including factors like sleep

What is the specific dynamic effect (SDE)?

Energy expenditure due to food digestion. Around 8-15 %

What is the formula to calculate TEO (total energy output)?

TEO = (BMR + ACT)(SDE)

What can be used to measure body frame size?

Getting calipers and measure sites with little overlying tissue, such as the wrists and elbows

How is a waist to hip ratio (WHR) determined?

Divide waist measurement by hip measurement

What does an apple shape look like for women?

Body fat located in upper body, greater risk of heart disease and harder for conception.

How many kcal per gram of: - Fat - Carbohydrates - Protein

- Fat (9) - Carbohydrates (4) - Protein (4)

What type of body fat measurement is best for people under 15% bf?

Skin fold calipers