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Flashcards in Lab 2 - Proteins Deck (18):
1

Def. on proteins

Large molecules, found in every cell, with essential importance in life processes. Contain amino acids.

2

Different structures of proteins

Structure elements, enzymes, transport and hormones

3

Terminology of proteins

1: Amino acid
2: Dipeptid
3-10: Oligopeptide
11-100: polupeptide
>100: protein

4

Structural levels

-Prim.: sequence of 10 proteinogen amino acids
-Sec.: conformation of peptide bonds (alfa-helix/beta-sheet, H-bond)
-Tert.: Conformation of peptide backbone+side chains+bonds/interactions betw. side chains (S-S, ionic, H-bond, hydrophobe interaction)
-Quert.: More than one polypeptide chain (myoglobin, haemaglobin)

5

Types of investigation

1.Qualitative(+/-): Denauturation-sulphosylic acid test, , renauturation, Biuret
2.Quantitative(conc.): Biuret, Tolin´s, refractometry
3.Separation(charachteristic): Salting out, gel-filtration, electrophoresis, dialysis(purification)

6

Sulphosylic acid test

Detection of prteinuria-white ppt. Precipitation(denaturation) of proteins by acids. Dissolved globular proteins changes into insoluble fibrous molecules. Either nitric acid, sulphosylic acid or tricholoroacetic acid.

7

Materials of sulphosylic acid test

Diluted blood plasma, sulphosylic acid, NaHCO3, pasteur pipette

8

Biuret reaction: name based on?

That biuret(two mol. of urea) gives same colour-R. as polypeptides and proteins.

9

Biuret reaction-qualitative

Detects poly-/oligopeptides of smaller mol. as well. Principle is that in alkaline milieu, the Cu2+ reacts with peptide bonds and form a violt-coloured complex. Should contain at least 2 peptide bonds.

10

Materials of biuret reaction-qualitative

Diluted blood plasma, NaOH sol., CuSO4 sol., pasteur pipette

11

Biuret reaction-quantitative

The proportion of sample and reagents required is very important. CuSO4 has to be in excess-for the intensity of colour - protein conc. betw. 200-400mg/l.

12

Materials of quantitative Biuret R.

-Diluted blood plasma
-Biuret reagent: dilute stock sol.; Segnette-salt(pot.sod.tartarate), NaOH, anhydrous CuSO4 and KI, with the diluent sol.; KI and NaOH
-Water bath at 37 degrees
-Spectrophotometer

13

Difference between qualitative and quantitative Biuret R.

-In the qualitative Cu2+ must be in excess (peptide bonds)-clear sol. is needed(photometry). Seignette salt is used to keep free Cu2+ in sol.
-The Biuret reagent is different.

14

Folin´s method-quantitative(by phenol reagent)

Two important proteins constituents: amino acid tyrosine and tryptophan, R. with Folin´s reagent. Blue colour formed due to reduction of phosphomolybdate and -tungstate into molybdateblue and tungstadblue. Can only be used with standard series of dilution only, and if protein conc. is betw. 200-500mg in 1L of protein sol.

15

Materias in Folin´s method-quantitative

-Diluted blood plasma
-Folin´s reagent |: Stock sol.; A.CuSO4, B.Seignette salt, C.Na2CO3
-Folin´s reagent ||: Of phosphoric tungstate, phosporic molybdate and litium sulphate in HCl.
-NaOH sol.
-Water bath at 37 degrees
-Spectrophotometer

16

Materials for salting out

-Blood plasma
-(NH4)2SO4
-Plastic funnel
-Filter paper

17

Salting out

Reversible denauturation. Hydrate hull keeps the proteins in solution, and can be drawn away by certains salts. Different salt conc. is needed. Increasing it gradually separates the protein.

18

What does the velocity depend on in gel electrophoresis?

-Intensity of the applied current-stable
-Medium-stable
-pH of the buffer-stable
-Electrophoretic index of this protein, which depends on change, shape and mol.mass.