1 Laboratory Activity Six

Introduction to the theory, concerns & applications in the handling of proteins for biochemical studies. Specifically:  Tissue disruption & protein extraction.  Salting in vs. salting out & ammonium sulfate fractionation.  Treatments that promote protein denaturation  Treatments that promote protein “protection”.  Methods for protein quantification. Introduction to the theory, concerns & applications in the handling of proteins for biochemical studies. Specifically:  Tissue disruption & protein extraction.  Salting in vs. salting out & ammonium sulfate fractionation.  Treatments that promote protein denaturation  Treatments that promote protein “protection”.  Methods for protein quantification. 2 2

 Proteins are most stable & functional in their native environment (i.e. the cells & tissues in which they are found).  pH = 7.4  Osmolality = 0.3 – 0.4  Compartmentalized.  Reducing environment.  Many biochemical studies involve removal/purification of proteins from their cellular environments.  Overall concern is to keep isolated proteins in their most functional condition during handling and studies.  Proteins are most stable & functional in their native environment (i.e. the cells & tissues in which they are found).  pH = 7.4  Osmolality = 0.3 – 0.4  Compartmentalized.  Reducing environment.  Many biochemical studies involve removal/purification of proteins from their cellular environments.  Overall concern is to keep isolated proteins in their most functional condition during handling and studies. 3 3

Important Concerns When Studying & Handling Proteins:  Choice of tissue disruption method to release protein(s) of interest.  Protection of protein extracts & components from endogenous & exogenous factors (i.e. maintenance of protein integrity).  Concentration & purification of individual protein components.  Estimation of protein concentrations / amounts.  Diversity of protein structures & functions. Important Concerns When Studying & Handling Proteins:  Choice of tissue disruption method to release protein(s) of interest.  Protection of protein extracts & components from endogenous & exogenous factors (i.e. maintenance of protein integrity).  Concentration & purification of individual protein components.  Estimation of protein concentrations / amounts.  Diversity of protein structures & functions. 4 4

Main Concerns:  Sheer forces vs. tissue/cell disruption vs. organelle & protein damage/destruction.  Sample dilution into homogenization buffer; thermal denaturation.  Release of toxic cellular contents; exposure to oxidizing conditions. Main Concerns:  Sheer forces vs. tissue/cell disruption vs. organelle & protein damage/destruction.  Sample dilution into homogenization buffer; thermal denaturation.  Release of toxic cellular contents; exposure to oxidizing conditions. 5 5

 Hydrolytic enzymes of lysosomes & plant vacuoles.  Phenolics, pigments, acids, ions of vacuoles.  Hydrolytic enzymes of lysosomes & plant vacuoles.  Phenolics, pigments, acids, ions of vacuoles. 6 6 Polyvynylpolypyrrolidone(PVPP) Tannic Acid

 Buffers  Thiol Reagents  Protease Inhibitors  Osmoticants  Detergents  PVPP  Low (non-freezing) Temperatures  Others (lab manual)  Buffers  Thiol Reagents  Protease Inhibitors  Osmoticants  Detergents  PVPP  Low (non-freezing) Temperatures  Others (lab manual) 7 7 Collectively referred to as the homogenization buffer or the homogenization “cocktail”.

Cell-Free Extracts Often Require “Reconcentration”: “Reconcentration”:  Lyophilization (freeze-drying).  Reverse Osmosis.  Salting Out.  Centrifugation (lab 10). Cell-Free Extracts Often Require “Reconcentration”: “Reconcentration”:  Lyophilization (freeze-drying).  Reverse Osmosis.  Salting Out.  Centrifugation (lab 10). 8 8 Semi-PermeableMembrane AddedPressurePureSolvent Solute of Interest Small-scale Lyophilizer

Salting In & Out of Proteins 9 Solubility Salt Concentration Salting in Salting out Protein Molecules Cations: N(CH 3 ) 3 + > NH 4 + > K + > Li + > Mg 2+ > Ca 2+ > Al 3+ > guanidinium Anions: SO 4 2- > HPO 4 2- > CH 3 COO - > citrate > tartrate > F - > Cl - > Br - > I - > NO 3 - > ClO 4 - > SCN -

Some Important Notes:  NH 4 SO 4 is normally the salt of choice.  Relatively high solubility (  4.0M; 528 g/L; 52.8% w/v).  Concentrated solutions have low densities; do not interfere with protein sedimentation.  Concentrated solutions are anti-microbial.  Concentrated solutions protect most proteins against denaturation.  Different proteins have different solubility curves.  Differential salting out can be used for purification.  Salt concentrations are expressed as a % of saturation.  % Saturation ≠ % (w/w) or % (w/v).  100% saturation = 20C; 0C. Some Important Notes:  NH 4 SO 4 is normally the salt of choice.  Relatively high solubility (  4.0M; 528 g/L; 52.8% w/v).  Concentrated solutions have low densities; do not interfere with protein sedimentation.  Concentrated solutions are anti-microbial.  Concentrated solutions protect most proteins against denaturation.  Different proteins have different solubility curves.  Differential salting out can be used for purification.  Salt concentrations are expressed as a % of saturation.  % Saturation ≠ % (w/w) or % (w/v).  100% saturation = 20C; 0C. 10

Preparation of a Cell-free Extract from Spinach Leaves:

Defined as... The disruption (or unfolding) of tertiary & secondary protein structure that leads to loss of protein function. Often manifested as the formation of cloudy precipitates or flocculation of protein. Defined as... The disruption (or unfolding) of tertiary & secondary protein structure that leads to loss of protein function. Often manifested as the formation of cloudy precipitates or flocculation of protein. 12

NH 4 SO 4 Fractionation of Spinach Cell-free Extract: 13 TA’s UV-280 UV-280 Protein Assay + Save fractions for electrophoresis (in two weeks). Recovery or purification of Rubisco subunits. Recovery or purification of Rubisco subunits. 383 mL 450 mL 27 mL

 Based on absorbance of aromatic amino 280 nm.  [Protein], mg/mL = ABS 280 x 1.55 [  280  mL/(mg  cm)]. Advantages:  Simple, non-destructive.  Fairly sensitive (20µg/mL - 3 mg/mL). Disadvantages:  Contaminants (especially nucleic acids) also 280 nm.  Variable amounts of aromatic amino acids in various proteins.  Based on absorbance of aromatic amino 280 nm.  [Protein], mg/mL = ABS 280 x 1.55 [  280  mL/(mg  cm)]. Advantages:  Simple, non-destructive.  Fairly sensitive (20µg/mL - 3 mg/mL). Disadvantages:  Contaminants (especially nucleic acids) also 280 nm.  Variable amounts of aromatic amino acids in various proteins. 14 PheTyr Trp His

 Based on the quantitative colorimetric shift that occurs when “Coomassie Brilliant Blue G-250” binds with proteins.  Binding promoted by hydrophobic & electrostatic interactions.  Based on the quantitative colorimetric shift that occurs when “Coomassie Brilliant Blue G-250” binds with proteins.  Binding promoted by hydrophobic & electrostatic interactions H H H + Basic Amino Acids (in protein structure) Coomassie Brilliant Blue G-250 Colorimetric Reactions Anionic form bound to protein ( max = 610 nm) Cationic form ( max = 470 nm)

Advantages:  Simple, quick, sensitive assay (1 – 200 µg/mL).  Few interfering substances. Disadvantages:  Unstable color reaction.  Relatively high blanks (  0.400A).  Not perfectly linear (i.e. limited linearity).  Different proteins bind different amounts of dye. Advantages:  Simple, quick, sensitive assay (1 – 200 µg/mL).  Few interfering substances. Disadvantages:  Unstable color reaction.  Relatively high blanks (  0.400A).  Not perfectly linear (i.e. limited linearity).  Different proteins bind different amounts of dye. 16

17 Bradford Estimates of 10 mg/mL Solutions Protein Conc.* Pepsin4.1  -Globulin (rabbit)8.0 Lysozyme9.9 Histones15.8 BSA21.1 Cytochrome c25.3 (*Data from Appendix XII; standard was Bovine  -Globulin)

 A quantitative test for protein (1 – 10 mg/mL).  Based on a colorimetric shift from blue to purple ( max = 540 nm).  Involves the formation of a “tetra-dentate” Cu 2+ -protein complex.  A quantitative test for protein (1 – 10 mg/mL).  Based on a colorimetric shift from blue to purple ( max = 540 nm).  Involves the formation of a “tetra-dentate” Cu 2+ -protein complex. 18 Protein + Biuret Reagent NaOH, CuSO 4 & Na/K-Tartrate (Tetra-dentate Complex) (*Performed as part of Activity 2)

 TA’s prepare cell-free extract & NH 4 SO 4 cuts.  Students:  Test effects of various treatments on protein denaturation.  Quantify protein in various fractions via UV-280.  Quantify protein in various fractions via Bradford.  TA’s prepare cell-free extract & NH 4 SO 4 cuts.  Students:  Test effects of various treatments on protein denaturation.  Quantify protein in various fractions via UV-280.  Quantify protein in various fractions via Bradford. 19

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