HYDROGEN ION BUFFERS FOR BIOLOGICAL RESEARCH ABSTRACT: twelve new or little used hydrogen ion buffers covering

the range pK a = 6 !" # $ %" have been prepared and tested Ten are &witterionic a'ino acids( either )* substituted taurines or )*substituted glycines( and two are cationic pri'ary aliphatic a'ines All of the &witterionic buffers are better than conventional buffers in the +ill reaction and in the phosphorylation*coupled o,idation of succinate by bean 'itochondria Two of the &witterions( )*Tris-hydro,y'ethyl.*'ethyla'inoethanesulfonic acid and )* /*hydro,yethylpipera&ine*)0*/*ethanesulfonic acid( give particularly active and stable 'itochondrial preparations These two also give higher rates of protein synthesis in cell* free bacterial preparations than do tris*-hydro,i'ethyl.a'ino'ethane -Tris. or phosphate buffers

oo few substance suitable for use as hydrogen ion buffers between p+ 6 and $ have been accessible to biologist in the past As a result so'e very inappropriate buffers have been used in biological research These buffers have been bad because of inefficacy( undesired reactivity( or to,icity 1hosphate has poor buffering capacity above p+ 2 " it tends to precipitate 'ost polyvalent cations and it is either a 'etabolite or an inhibitor in 'any syste's Tris-hydro,y'ethyl.a'ino'ethane -Tris. has poor buffer ing capacity below p+ 2 " 'oreover it is a pri'ary aliphatic a'ine of considerable reactivity and conse3uently it is often inhibitory Borate is notorious for co'ple,ing a wide range of organic co'pounds n*cluding 'any respiratory inter'ediates 4lyclyglycine is an e,cellent buffer above p+ $ but it is al'ost useless below p+ 2 " it is also rather e,pensive 5ther buffers such as i'ida&ole( veronal -("* diethybarbiturate.( 'aleate( and di'ethyglutarate are for one reason or another even less satisfactory The inade3uacy of our inventory of buffers 'ay be a 'ore serious proble' than we reali&e 6e cannot assu'e that various particulate syste's and soluble en&y'e syste's all respond the sa'e way to different ph*stabili&ing addenda( and there 'ay be no one ideal buffer for given p+ range 7nfortunately the nu'ber of available buffers is so li'ited that we have been able to evaluate properly those we do have 8t is i'possible even to guess how 'any e,ploratory e,peri'ents have failed( how 'any reaction rates have been depressed( and how 'any processes have been distorted because of the i'perfections of the buffers e'ployed This situation is unnecessary Che'ist have long understood( at least in a 3ualitive way( the relation of 'olecular structure to the che'ical and physical properties( and prepare a wide variety of ine,pensive co'pounds with desired dissociation constants( solubilities( and reactivities The following is a report on the synthesis( properties( and utility of so'e new buffers 9any 'ore could be prepared( and we 'a:e no clai's as to the universal superiority of the particular substances described 8n designing the new buffers the following rules of 'erit have been accepted 6e reali&e( of course( that not all of these criteria are li:ely to be valid in all instances ! / The pK -i e ( the p+ of the 'idpoint of the buffering range. should be between 6 and $ since this is the region of fewest buffers and 'ost biological resotions The buffer should have 'a,i'u' never solubility and 'ini'u' solubility in all other solvents 4reat water solubility is convenient in that it per'its the use of concentrated buffer stoc:s 9uch 'ore i'portant( however( is the ratio of the

% < " 6 2 $ >

solubility in water to the solubility in relatively nopolar solvents since this deter'ines the distribution of buffer between the a3ueous 'ediu' and the biological phase in particulate syste's Thus( for a given amount of buffering the concentration of buffer inside the cellular organelles is lower with 'ore polo buffers ;or the sa'e reason -'ini'u' e,traneous 'aterial in the particles. the buffer should pass through biological 'e'branes with difficulty The buffers should produce a 'ini'u' of salt effects 8f the syste' to be studied re3uires salts( suitable ions can be added( but if the syste's is adversely affected by salt( ionic buffers create proble's There should be a 'ini'u' influence of buffer concentration( te'perature( and ionic co'position of the 'ediu' on the dissociation of the buffer co'ple,es for'ed with cations should be soluble and the binding constants of the co'ple,es should in :nown 8n the interest of si'plicity( so'e of the buffers should not 'a:e such co'ple,es the buffers should be as stable as possible They should resist en&y'atic and nonen&y'atic degradation under the conditions of use and they should not rese'ble en&y'e substrates to the e,tent of acong es analog inhibitors They should not absorb light in the visible or ultraviolet regions of the spectru' Absorption at wavelengths grater than /%=*/<= ' could interfere with widely used spectrophoto'etric assays They should be easily prepared fro' ine,pensive 'aterials and should be easily purified by si'ple procedures such as recrystalli&ation

So'e of these characteristics( e,tre'e polarity( reduced 'e'brane penetration( reduced ion effects( and crystalli&ability( are the characteristics of &witterions 5ther characteristics such as acid dissociation at and above neutrality( absence of ultraviolet absorption( and resistance to o,idation are characteristics of 'any secondary and tertiary aliphatic a'ines Conse3uently 'ost of the buffers described below are &witterionic aliphatic a'ines( that is to say a'ino acids The 'a?ority are either substituted glycines or substituted taurines 8t is not difficult to synthesi&e aliphatic a'ines with appropriate acid dissociation constants Si'ple al:yl*a'ines are protonated far too readily for our purpose and therefore they only buffer at very low hydrogen ion concentrations ;or instance( the pKa of ethyla'ine is != 2" and the pKa of glycine is > > +owever protonation beco'es 'ore difficult and the pKa is lowered accordingly under a variety of circu'stances Thus the presence of the electronegative a'ido group ad?acent to the nitrogen usually reduces the pKa by / " # % units Several hydro,yls -as in Tris or triethanola'ine. have a si'ilar effect Ad?acent for'al positive charges -as in half*protonated ethylenedia'ine. are responsible for an even larger lowering of the p:a about % " units The pKa is also greatly lowered in those tertiary a'ines where the nitrogen is in a ring( since the protonated fro' is then considerably strained and therefore less easily for'ed By ta:ing advantage of these inductive( electrostatic( and steric influences( either prepared with pKa values ranging fro' well below % to well aboye !! 8ncorporation of at least one anionic group provides us with &witterions capable of buffering in the desired p+ range Twelve new or little buffers are described below 8'portant physical and che'ical properties( i.e., 1:a( values at different te'peratures( 'etal*buffer binding constants(

solubilities in water( etc ( have been 'easured 6e have also evaluated the new buffers by co'paring the'( with standard buffers such as phosphate( Tris( and glycylglycine in three different biological syste' 5ur preli'inary tests suggest that several of the new buffers 'ay be superior to any of the buffers now widely e'ployed ater!als an" met#o"s Starting materials. Details of the syntheses of the buffer will be presented in a separate section The following is a list of all the starting 'aterials with the co''ercial source of each: chloroaceta'ide( chloroacetic acid( and bro'oacetic acid fro' @ast'an 5rganic che'icals( Rochester( ) A B 'orpholine( anhydrous tri'ethyla'ine( Tris-hydro,y'ethyl.a'ino'athane( diethanola'ine( and glycine fro' ;isher Scientific Co ( ;airlawn( ) C B anhydrous sodiu' sulfite and concentrated a3ueous a''onia fro' C T Ba:er Che'ical Co ( 1hillipsburg( ) C B )*/*hydro,yethylpipera&ine fro' Aldrich Che'ical Co ( 9ilwau:ee( wis B pipera&ine he,ahydarte fro' )utritional Bioche'icals Corp ( Cleveland( 5hioB !(/*dibro'oethane fro' 9atheson Cole'an and Bell( )orwood( 5hio Sodiu' /*bro'o*etanesulfonate was prepared by the 'ethod of 9arvel and Sparberg -!><%. Acid Dissociation Constants. Dissociation constants were deter'ined by titrating the new buffers with a Radio'eter auto'atic tirator*titrigraph which had been standardi&ed by titrating well*:nown buffers under the sa'e conditions 1Ka values were 'easured at several concentrations and several te'peratures )eutrali&ation e3uivalents were deter'ined at the sa'e ti'e with an error of about E !F Metal Binding Constants. Appro,i'ate values for the 'etal*buffer binding constants were deduced fro' the displace'ent of the p+ titration curve in the presence of an e3uivalent of the chloride salt of the 'etal in 3uestion 8f we assu'e that the 'etal fro's a coordinate bond with the a'ine nitrogen of the buffer and in so doing co'petes with protons( the following e3uations e,press the e3uilibriu' condition GAH = G)H I G)+IH I G)9IH -!.

where GAH is the total buffer concentration ( G)H is the concentration of free a'ine( G)+IH is the concentration of protonated a'ine( and G)9IH is the concentration of 'etal* buffer co'ple,( obviously also G9H = G9/IH I G)9IH -/.

where G9H is the total 'etal concentration and G9/IH is the concentration of free 'etal ion @ach of the two co'peting processes has its own e3uilibriu' e3uation Thus Ka = G)HG+IH J G)+IH -%. 6here Ka is the acid dissociation constant of the buffer and K' = G)9IH J G)H G'/IH -<.

6here K' is the 'etal*buffer binding constant By substituting e3 !( / and % in e3 <( we obtain -". GAH # KaG)+IH J G+IH # G)+IH -KaG)+IH J G+IH. -G9H # GAH I KaG)+IH J G+IH I G)+IH. 1resu'ably all of the buffer is in the protonated fro' at the beginning of the titration( and therefore it is reasonable to assu'e that half is in the protonated fro' at the 'idpoint of the titration curve( or G)+IH = GAH J / ;urther'ore( in our e,peri'ents GAH = G9H Thus for the 'idpoint of the titration curve e3 " reduces to K' = / -G+IH J Ka # !. GAH-KaG+IH I !. -6.

The three values( G+IH( Ka( and GAH( are provided by the titration curves observed in the presence and absence of the 'etal Co'ple, for'ation with 'agnesiu'( calciu'( 'anganous( and cupric ions was investigated in this 'anner 6hen we report no 'etal binding we refer only to these four 'etals Other properties. The 'elting points or deco'position points of the buffers were deter'ined on a ;ischer*Chons 'elting point bloc: which had been calibrated with :nown substances Solubilities of the buffers at =K were deter'ined by titrating ali3uots of saturated solutions 7ltraviolet spectra of = =" 9 solutions of the buffers were deter'ined by a Bausch and Lo'b Spectronic "=" spectrophoto'eter Biological Test. Reduction of potassiu' ferricyanide by illu'inated chloroplasts - the +ill reaction. was 'easured spectrophoto'etrically by the 'ethod of 8&awa and 4ood -!>6". 5,idation of succinate by bean hypocotyl 'itochondria - Phaseolus vulgaris L.) was 'easured as o,ygen upta:e in 6arburg vessels at /=K 1rotein synthesis in cell*free e,tracts of scherichia coli was 'easured as incorporation of radioactive L*leucine Details of the procedures are given in the @,peri'ental Section @M1@R89@)TAL S@CT85) Preparation and properties o! the "u!!ers tested. /*-)*9orpholino.ethanesulfonic acid -9al:iel and 'ason( !><%. was prepared by reflu,ing an a3uous solution of sodiu' bro'oethanesulfonate with a large e,cess of 'orpholine for /hr The greater part of the unreacted 'orpholine was re'oved by distillation at reduced pressure and the residue was ta:en up in water Sodiu' ions and residual 'orpholine were re'oved by passing the resulting solution through a sulfonic acid resin colu'n -Dowe, "=. in the acid fro' The eluate( which contained pri'arily product and hydrogen bro'ide( was ta:en al'ost to dryness by distillation at reduce pressure The faintly colored product which precipitated on addition of alcohol was redissolved in hot alcohol containing a little water( decolori&ed with )orit( and recrystalli&ed coolingB yield $"F A second recrystalli&ation fro' alcohol and water gave a colorless 'aterial which deco'posed above %==KB apparent pKa when = !9: =K 6 %$B /=K( 6 !"B %2K( " >$B 'etal*buffer binding constant -log Ka. at /=K( and = !9: 9g /I( = $B Ca/I( = 2B 9n/I( = 2B cu/I( negligibleB effect of concentration on the apparent pKa at =K: = /9( 6 !2B = =! 9( 6 !$B concentration of a satured a3ueous solution at =K( = 6"9B proposed trivial na'e( 9@S Anal. Caled: )( 2 !$B neut e3uiv( !>" ;ound: ) -K?eldahl.( 2=6B neut e3uiv( !>" E /

)*-/*aceta'ido.i'inodiacetic acid -Schwar&en*bach et al.( !>"". was prepared by the reaction of / e3uiv of sodiu' bro'o acetate with ! e3uiv of glycina'ide hydrochloride -see below. in water A concentrated solution of )a5+ was added at roo' te'perature with stirring at a rate which 'aintained the p+ between 2 and $ after about ! " hr % e3uiv of )a5+ had been added and the p+ was steady at $ The reaction 'i,ture was then acidified to p+ / / with +CL( whereupon the very insoluble free acid precipitated and the p+ rose The 'other li3uors were again acidified to p+ / = and a second crop of the free acid precipitated The product was purified by converting it into the very soluble 'onosodiu' sait and reprecipitating the free acidB yield -fro' chloroaceta'ideB see glycina'ide below. ""FB deco'p pt //=KB pKa ! below /B pKa/ probably between / and %B apparent pKa% when = !9: =K( 6 $"B /=K( 6 6/B %2K( 6 <"B 'etal*buffer binding constants -log K$. as cited by Schwar&eribach et al. -!>"".: effect of concentration on apparent pKa at /=K: = !"9( 6 6=B = =!9( 6 2$B solubility of free acid very s'allB solubility in buffering range -'ono* nad disodiu' salts. very highB proposed trivial na'e( ADA Anal. Calcd: neut e3uiv( >" ;ound: neut e3uiv >" E ! 181@RAN8)@*)()0* B8S -/*@T+A)@S7L;5)8C AC8D. was prepared by reflu,ing a solution containing / e%u!& of sodiu' bro'oethanesulfonate with ! e3uiv of pipera&ine he,ahydrate for /hr After = " hr a concentrated solution of )a5+ was added dropwise at a rate which :ept the p+ of cooled( diluted ali3uots between > and != 6hen / e3uiv of al:ali had been added and no 'ore was being consu'ed( the reaction 'i,ture was cooled and acidified to p+ ! " with +CL The very insoluble double &wittrion i''ediately precipitatedB yield 6=F A s'all portion was recrystalli&ed fro' boiling water -solubility about ! g l' at !==K. The greater part was purified by repeated conversions to the very soluble 'onosodiu' salt and reprecipitations of the acid Another portion was converted to the 'ost 'onosodiu' salt and recrystalli&ed fro' water and alcoholB deco'p pt above %==KB pKa! below%B apparent p:a/ when = !9: =K( 2 =/B /=K( 6 $/B %2K( 6 2=B no 'etal bindingB effect of concentration on the apparent pKa/ at /=K: = /9( 6 $/B = =!9( 6 >6B solubility in buffering range -'ono*and disodiu' salts. very highB proposed trivial na'e( B@S Anal. Calcd: > /2B neut e3uiv( !"! ;ound: ) -K?eldahl.( > /= neut e3uiv( !<> " E ! " )*-/*AC@TA98D5.*/*A98)5@T+A)@S7L;5)8C AC8D was prepared by reflu,ing an a3ueous solution containing ! e3uiv of sodiu' bro'oethanesulfonate with ! e3uiv of glycina'ide hydrochloride - see below. A concentrated solution of )a5+ was added dropwise at a rate which :ept the p+ of cooled and diluted ali3uots at about $ % After / e3uiv of )a5+ had been added and no 'ore al:ali was consu'ed( the reaction 'i,ture was cooled and acidified to p+ % with +CL 5n addition of one volu'e of alcohol the product precipitatedB yield "=F After recrystalli&ation fro' hot water and alcohol( the product deco'posed at />%KB apparent pKa when = !9: =K( 2 %/B /=K( 6 $$B %2K( 6 "6B 'etal*buffer binding constants -log K'. at /=K and = !9: 9g /I( = <B Ca/I( = <B 9n/I( negligibleB Cu/I( < ">B effect of concentration on the apparent pKa when = !9( 6 >=B concentration of a saturated a3ueous solution at =K( = //9B proposed trivial na'e( AC@S

Anal. Calcd: )( !" %$ neut e3uiv( !$/ ==/ -K?eldahl.( !" /6B neut e3uiv( !$/=/ -/*A98)5@T+AL.TR89@T+ALA995)879 C+L5R8D@ +ADR5C+L5R8D@ -4abriel( !>/=. was prepared by the action of e,cess concentrated a3ueous a''onia on -/* bro'oethyl. tri'ethyla''oniu' bro'ide followed by an e,changue of bro'ide ions for chioride ions on an a'ine resin n the chloride for' The -/*bro'oethyl. tri'ethyla''oniu' bro'ide was prepared in good yield by the action of ! e3uiv of anhydrous tri'ethyla'ine on ! e3uiv of !(/*dibro'oethane in nitroben&ene After recrystalli&ation fro' alcohol( ! e3uiv of the -/*bro'oethy.tri'ethyla''oniu' salt was dissolved in !== e3uiv of concentrated a3ueous a''onia After standing for ! wee: at roo' te'perature( the solution was ta:en to dryness by distillation at reduced pressure The residue was recrystalli&ed fro' alcohol( redissolved in water( and passed over a large a'ount of a'ine resin -Dowe, %. in the chloride fro' The eluate was again ta:en to dryness and the residue was recrystalli&ed fro' alcoholB deco'p pt /6=KB apparent pKa when = !9: =K 2 6$B /=K( 2 !=B %2K 6 6$B no 'etal biding affect of concentration on apparent pKa at /=K: = /9( 2 !$B = =!9( 6 $$B concentration of a saturated a3ueous solution at =K( < /9B trivial na'e fro' the literature( Chola'ine Anal. Calcd: neut e3uiv( !2" ;ound: neut e3uiv !2" E / )()*Brs -/*+ADR5MA@T+AL.*/*A98)5@T+A)@S7L;5)8C AC8D -8&u'i( !>"<. was prepared by reflu,ing an a3ueous solution of sodiu' bro'oethanesulfonate with / e3uiv of diethanola'ine for / hr The cooled reaction 'i,ture was passed over a sulfonic acid resin -Dowe,"=.in the acid fro' The elaute( containing product and +Br( was ta:en to dryness at reduced pressure and the product was recrystalli&ed fro' a3ueous alcoholB yield "/FB 'p !"%*!""KB apparent pKa when = !9: 9g /I( Ca/I( and 9n/I( negligibleB Cu/I( % "=B effect of concentration on the apparent pKa at /=K: = /9( 2 !$B = =!9( 2 /=Bconcentration of a saturated a3ueous solution at =K( % /9B proposed trivial na'e( B@S Anal. Calcd: )( 6 "2B neut e3uiv( /!% ;ound: )( -K?eldahl.( 6 "<B neut e3uiv( /!! E / #*Tris-+ADR5MA9@T+AL.9@T+AL*/*A98)5@T+A)@S7L;5)8C AC8D was prepared by reflu,ing an a3ueous solution of sodiu' bro'oethanesulfonate with ! " e3uiv of tris$+ADR5MA9@T+AL.a'ino'ethane -Tris. for / hr The cooled reaction 'i,ture was passed over a sulfonic acid resin -Dowe, "=. in he acid fro' and the eluate was concentrated to a very s'all volu'e by distillation at reduced pressure The product precipitated on addition at reduced pressure 8t was redissolved in hot alcohol containing a little water and recrystalli&ed by coolingB yield 6=FB 'p //6*//$KB deco'p pt /%!KB apparent pKa when = !9: =K( 2 >/B /=K( 2 "=B %2K( 2 !<B 'etal*buffer binding constants $log K'. at /=K and = !9: 9g/I( Ca/I( and 9n/I( negligibleB Cu/I( % $=B effect of concentration on the apparent pKa at /=K: = /9( 2 "=B = =!9( 2 "<B concentration of a saturated a3ueous solution at =K( / 69B proposed trivial na'e( T@S Anal. Calcd: )( 6 !! neut e3uiv( //> ;ound: )( -K?eldahl.( 6 =>B neut e3uiv( //> E / )*/*+ADR5MA@TCAL181@RAN8)@*)0*/*@T+A)@S7L;5)8C AC8D 6as prepared by reflu,ing an a3ueous solution of sodiu' bro'oethanesulfonate with ! e3uiv of )*/*hydro,yeethylpipera&ine for /hr after = "hr a concentrated solution of )a5+ had been added at a rate which :ept the p+ of cooled and diluted ali3uots at about > = 6hen about ! e3uiv of )a5+ had been added and there was no further consu'ption of al:ali( the reaction 'i,ture was cooled and poured through a sulfonic acid resin -Dowe, "=. colu'n in the acid fro'

The colu'n was washed thoroughly with distilled water and then eluted with an e,cess of dilute a3ueous a''onia Al'ost pure product ca'e off the colu'n as soon as the sulfonic acid of the resin was neutrali&ed The eluate was evaporated to dryness at reduced pressure( and the product was suspended in alcohol and a little water 4lacial acetic acid was added until ali3uots of this suspension diluted with water were at p+ " =( the isoelectric point of the product The suspension was then cooled in an ice bath( filtered( redissolved in hot alcohol and water( and reprecipitated by cooling B yield 2"FB 'p /%<KB pKa! about %B apparent pKa/ when = !9: =K( 2 $"B /=K( 2 ""B %2K( 2 %!B no 'etal bindingB effect of concentration on the apparent pKa /at /=K: = /9( 2""B = =!9( 2 "": concentration of a saturated a3ueous solution at =K( / /"9B proposed trivial na'e( +@1@S Anal. Calcd: )( !! 2"B neut e3uiv -second acid dissociation only. /%$ ;ound: ) -K?eldahl.( !! 6/B neut e3uiv( /%$ E / )*-/*AC@TA98D5.4LAC8)@ was prepared by the action of sodiu' glycinate on chloroacte'ide 4lycine -/e3uiv. and ! e3uiv of )a5+ were dissolved in a very s'all volu'e of water at roo' te'perature and !e3uiv 5f chloroaceta'ide was stirred in A concentrated solution of )a5+ was added at a rate which 'aintained the p+ at != The te'perature was not per'itted to rise above <=K After /*%hr( depending on the te'perature( slightly 'ore than ! e3uiv of )a5+ had been added and the chloroaceta'ide had all dissolved Titration of the reaction 'i,ture showed an $"F yield of a substance with pKa about 2 2 Acidification of the reaction 'i,ture to p+ " = with acetic acid( re'oval of 'ost of the water( and additions of successive a'ounts of alcohol yielded first the e,cess glycine and then the e,tre'ely soluble product badly conta'inated with glycine Recrystalli&ation resulted in a preparation which was $=F product and /=F glycine Since we have neither developed a practical synthesis of aceta'idoglycine nor obtained a pure sa'ple( we report here only its appro,i'ate pKa/ and the fact that is shows so'e pro'ise as a buffer for 'itochondrial suspensions #*Tris-+ADR5MA9@T+AL.9@T+AL4LAC8)@ was prepared by the 'ethod previously described -4ood( !>6/.B pKa! appro,i'ately / %B apparent pKa/ when = !9: =K( $ 6B /=K( $ !"B %2K( 2 $B 'etal*buffer binding constants -Log K'. at /=K and = !9: 9g/I( ! /B Ca/I( / <B 9n/I( / 2B Cu/I( 2 %B effect of concentration on the apparent pKa/ at /=K: = /9( $ !"B = =!9( $ !"B concentration of a saturated a3ueous solution at =K( < 69 #*Tris-+ADR5MA9@T+AL. A98)5@T+A)@ for biological research -Tri&'a base. was obtained fro' Sig'a Che'ical Corp ( St Louis( 9o B apparent pKa when = !9: =K( $ >B /=K( $ %B %2K( 2 >B negligible 'etal bindingB effect of concentration on the apparent pKa at /=K: = /9( $ %B = =!9( $ /B concentration of a saturated a3ueous solution at =K( ! !9B trivial na'e widely e'ployed in the literature( Tris #*#*Bis-/*+ADR5MA9@T+AL.4LAC8)@ -Kiprianov( !>6/. was prepared by the action of e,cess diethanola'ine on bro'oacetic acid in water The product separated in good yield on the addition of alcohol to the reaction 'i,tureB apparent pKa! when = !9: =K( $ 2B /=K( $ %"B %2K( $ /B ('etal*buffer binding constants -Log K'. when = !9: 9g /I( ! "B Ca/I( / $B 9n/I( % !B Cu/I( $ !B effect of concentration on the apparent pKa/ at /=K: = /9( $ %B = =!9( $ < concentration of saturated a3ueous solution at *******9B proposed trivial na'e( Bicine The use of this co'pound as a buffer for biological investigation has been suggested by Re'i&ov -!>6=.

4LACAL4LAC8)@ was prepared by the action of a large e,cess of a3ueous a''onia on chloroacetylglycine -Cheronis and Spit&'ueller( !><!.B apparent pKa / when = !9: =K( > =B /=K( $ <B %2K( 2 >B 'etal*buffer binding constants -Log K'. at /=K and = !: 9g /I( = $B Ca/I( = $B 9n/I( ! 2B Cu/I( " $B effect of concentration on the apparent pKa/ at /=K: = /9( $ <B = =!9( $ <B concentration of a saturated a3ueous solution at =K( ! !9 At buffering concentrations -= ="9.( our preparations of 9@S( 181@S( Chola'ine( B@S( T@S( +@1@S( TricineB and Bicine absorbed a negligible a'ount of light at wavelengths greater than /<=' and very little light at %/=' AC@S absorbed significant a'ounts of light at /%=' -5D= = 2%. while our preparation of ADA absorbed light at wavelengths up to /6=' -5D= = /!. To the best of our :nowledge pipera&ine )()0*Bis-/*ethanesulfonic acid. -181@S.( )*-/*aceta'ido.*/* a'inoethanesulfonic acid -AC@S.( )*Tris*-hydro,y'ethyl.'ethyl*/* a'inoethanelsulfonic acid -T@S.( )*/*hydro,yethylpipera&ine*)0*/*ethanesulfonic acid -+@1@S.( and )*-/*aceta'ido.glycine have not been described before 6e wish to e'phasi&e that the data describing the physical properties of the buffers -su''ari&ed in table 8. have been selected on the basis of usefulness to biologists The scope and accuracy of these data are not such as to co''end our wor: to physical che'ist( not are our results couched in the ther'odyna'ical ter's they find convenientB rather we have presented the data with an eye to co''on bioche'ical procedures T$ $%LL & ACT%O# '%T$ (%(T # D%(( & #T B)(( &S. ( resh leaves of spinach -spinacia oleracea L . were ho'ogeni&ed in a waring Blendor for != sec at =K in a 'ediu' containing = %"9 )aCl(= ="9 sodiu' phosphate( and !'9 @DTA( p+ 2 % The ho'ogenate was filtered through three layers of cheesecloth and centrifuged at !"==g for " 'in The pellet was suspended in a 'ediu' containing = !"9 sucrose and = ="9 Tricine*)ao+( p+ 2 < After a brief centrifugation to re'ove cell debris( the chloroplasts in a s'all a'ount of the sucrose*Tricine buffer +ill reaction rates were 'easured by following the reduction of potassiu' ferricyanade in a 'odified Bausch and Lo'b Spectronic "=" spectrophoto'eter Absorbance changes at </=' were recorded on a strip*char recorder Actinic light of saturating intensity fro' a "==w pro?ector was passed through a red glass filter -trans'ission O6==' . the te'perature was !"K Reaction 'i,tures -/'l. consisted of chloroplasts containing %= g of chlorophyll( ! 6'oles of potassiu' ferricynade( the indicated a'ounts of the carious buffers at p+ 2 <( and s'all a'ounts of Tricine -" 'oles. and sucrose -!" 'oles. introduced with the chloroplasts ;or the 'easure'ent of electron transport uncoupled fro' phosphorylation( 'ethyla'ine hydrochloride -!=='oles. was added ;847R@!: RAT@S 5; ;@RR8CA)AD@ R@D7CT85) BA 8LL798)AT@D S18)AC+ C+L5R51LASTS 68T+ D8;;@R@)T B7;;@RS R@ACT85) 98MT7R@: C+L5R51LASTS C5)TA8)8)4 %= 4 5; C+L5R51+ALL( 8)D8CAT@D A957)T 5; T+@ PAR857S B7;;@RS AT 1+ 2 <( "95L@S 5; TR8C8)@ A)D !" 95L@S 5; S7CR5S@ 8)TR5D7C@D ;R59 T+@ C+L5R51LAST ST5CK S7S1@)S85)( ! 6 95L@S 5; 15TASS879 ;@RR8CA)AD@( ;8)AL P5L79@ / 9LB L84+T SAT7RAT8)4B T@91@RAT7R@ !"KB S5L8D L8)@S( !== 95L@S 5; 9@T+ALA98)@ +ADR5C+L5R8D@ T5 7)C571L@ @L@CTR5)

TRA)S15RT ;R59 1+5S1+5RALAT85)B BR5K@) L8)@S( )5 9@T+ALA98)@ T+@ 9AM8979 RAT@ 5; ;@RR8CA)AD@ R@D7CT85) -!== 5) ;847R@S. 6AS AB57T !===95L@SJ+R 1@R 94 5; C+L5R51+ALL ALL RAT@S AR@ C591AR@D T5 T+@ RAT@ 68T+ = ="9 TR8C8)@ S8)C@ )5 C5)C@)TRAT85) 5; A)A 5T+@R B7;;@R 4AP@ +84+@R RAT@S )5T@ T+AT ALL 5; T+@ N68TT@R85)8C B7;;@RS 7)C571L@ A)D 8)+8B8T L@SS T+A) @8T+@R T+@ CAT85)8C 5R T+@ A)85)8C B7;;@RS ;igures !( / and % are largely self*e,planatory The high rates -solid lines. are the 'ethyla'ine*uncoupled rates and the low rates -bro:en lines. are the basal rates A decrease in one of the high rates is presu'ably an e,pression of uncoupling Clearly all of the buffers at concentrations above = !9 inhibit the uncoupled rate and 'any uncoupled the basal rate +owever( within these general si'ilarities there are stri:ing differences The three &witterionic*substituted glycines -4lycylglycine( Tricine( and Bicine. inhibit to the sa'e e,tent as sucrose and therefore we 'ay assu'e that they inhibit solely by adversely affecting the os'olarity of the 'ediu' These three have little or no uncoupling effect at any concentration 8n contrast( the three cationic pri'ary a'ines -Tris( Chola'ine( and glycina'ide. are disastrous at high concentrationsB glycina'ide is an uncouple rivaling 'ethyla'ine in potency( while at slightly higher concentrations it is an effective inhibitor The substituted taurines -9@S( AC@S( B@S( T@S( and +@1@S. are inter'ediate So'e are slightly 'ore inhibitory in the uncoupled syste' than the substituted glycines or sucrose and all have slight uncoupling effects at high concentrations 8t should be noted( however( that these taurines have lower pKa values than the glycines and conse3uently a 'uch higher proportion of the buffer is in the anionic for'at p+ 2 < -The glycines with pKa values above $ are overwhel'ingly in the &witterionic for' at p+ 2 < . The sy''etrical disulfonic acid( 181@S( see's to be a rather effective uncouple at high concentrations but this 'ay reflect the presence of i'purities in a not easily recrystalli&ed substance The anionic buffers( phosphate and 'aleate( uncouple slightly 'ore than Tris( but are less inhibitory at high concentrations -Bicine has the disadvantage for +ill reaction studies that it is slowly o,idi&ed by ferricynade at p+ 2 < . 6e 'ay su' up the co'parison of the effects of the buffers on the +ill reaction as follows All of the &witterionic buffers are better than ionic buffers such as Tris( phosphate( etc ( and the 'ore of the buffer in the &witterionic for' the better it is This applies both to the inhibition of already uncoupled electron transport and to the uncoupling of electron transport The cationic pri'ary a'ine buffers inhibit the uncoupled rate and uncouple the coupled syste' The anionic buffers( as shown previously -4ood( !>6/.( are uncouplers of photophosphorylation ;847R@/: RAT@S 5; ;@RR8CA)AD@ R@D7CT85) S@@ CA1T85) T5 ;847R@ ! ;5R C5)D8T85)S

P$OSP$O&*LAT%O#+CO)MPL D O,%DAT%O# O( S)CC%#AT B* B A# M%TOC$O#D&%A '%T$ (%(T # D%(( & #T B)(( &S. @tiolated hypocotyls of pinto beans -1haseolus vulgaris L . were ho'ogeni&ed in a warning Blendor for !" sec -$=P. in the following 'ediu': sucrose = /"9 @DTA = =!9( bovine seru' albu'in -fraction P powder( 1ente, 8nc ( Kan:a:ee( 888 . = "F and the indicated buffer = ="9( p+ 2 < 1ellets were washed with the sa'e 'ediu' lac:ing @DTA The centrifugation

procedure was a 'odified version of that of Lyons et al -!>6<.: low speed -/===g for " 'in.( pellet discardedB high speed -!2 %==g for != 'in.( supernatant discardedB another high speed( supernatant discardedB low speed -!2 %==g for != 'in.( pellet discardedB another high speed( supernatant discarded The final pellet was suspended in /*% 'l of = /"9 sucrose 5,ygen upta:e was 'easured in 6arburg 'ono'eters and phosphorylation was 'easured as disappearance of orthophosphate The reaction 'i,ture -%'l. consisted of <"=' of sucrose( 6='oles of @DTA( = " 'g of he,o:inase -)utrional Bioche'ical Corp .( 6='oles of glucose( = ! 'g of cytochro'e c( /= 'g of bovine seru' albu'in -if used.( = %'ole of AD1( = % ''oles of AT1( 6= 'oles of sodiu' succinate( 6= 'oles -= =/9. of the indicated buffer( p+ 2 <( and 'itochondria containing between = % and = /'g of protein The te'perature was /=K ;irst( a preli'inary screening of the buffers was underta:en @ach buffer was placed in a different vessel Then the re'aining co'ponents of the 'ediu' and 'itochondria fro' a co''on stoc: were added 8n this study 'itochondria had been prepared by grinding and centrifuging in Tricine buffer with seru' albu'in @very buffer tested with the e,ception of 'aleate was better than Tris This is not a 'easure of the e,cellent of the buffers but of the shortco'ings of Tris since the reaction 'i,tures already contained phosphate( and phosphate alone gave rates %"F higher than Tris plus phosphate The conclusion to be draw fro' the first colu'n of results in table 88 is that 'ost of these buffers are innocuous but superfluous in the already buffered 'ediu' A few( such as ADA( AC@S( and Chola'ine( 'ay be slightly inhibitory -'uch lesson than Tris.( whereas a few( such as T@S( +@1@S( aceta'idoglycine( and 4lycylglycine( see' to be beneficial The second colu'n of table 88 shows that seru' albu'in can protect the 'itochondria( to so'e e,tent at least( fro' the har'ful effects of Tris. are higher than those in the first colu'n( pri'arily because the reates with Tris were lower +owever( the rates with +@1@S and T@S were( an absolute basis( higher in the absence of seru' albu'in than in its presence This raises again the interesting and as yet unsettled 3uestion of the nature of the 'itochondrial deterioration which seru' albu'in arrests 6hen we had established by these si'ple tests which of the new buffers showed pro'ise( we began the 'uch 'ore laborious tas: of co'paring rates of o,ygen upta:e and phosphorylation when the 'itochondria had been isolated and stored in the selected buffers 8n these e,peri'ents two new factors enter the picture: -a. the 'itochondria are e,posed fro' the first to the condition we are interested in( and therefore we should e,pect to find the already observed differences e,aggeratedB -b. bean hypocotyls contain an appreciable a'ount of acid and thus buffering capacity of the buffers at p+ 2 < is critical( the values in the third colu'n of table 88 reflect both the buffering efficiency and the other 'erits of the buffer 9@S fails 'iserably -being al'ost as bad as Tris. because( at p+ 2 <( it buffers so poorly( while the otherwise e,cellent Bicine is even worse than Tris because it co'pletely fails to neutrali&e the acid released when the tissues are ho'ogeni&ed T@S and +@1@S are outstanding( partly because they buffer so well -pKa values =2 " and 2 "". and partly for other reason we do not yet understand The 1J5 ratios averaged about ! < in these e,peri'ents 1hosphorylation efficiency did not vary consistently with rates of electron transport or with the different buffers 9oreover( none of the buffers caused a significant increase in the low rate of o,ygen upta:e in the absence of succinate Conse3uently( the higher rates of electron transport encountered with so'e of the buffers cannot be attributed to uncoupling or to buffer o,idation

;847R@ < shows the ti'e course of succinate o,idation when the 'itochondria were prepared and the reactions carried out in five different buffers +@1@S and T@S see' to have positive protective actions beyond their buffering functions %#CO&PO&AT%O# O( -./C0L+L )C%# into T&%C$LO&OAC T%C AC%D P& C%P%TABL P&OT %# '%T$ (O)& D%(( & #T B)(( &S. . coli cells growing e,ponentially in a salt* glucose 'ediu' supple'ented with L*histidine( L* leucine( and uracil were harvested and washed in a buffer consisting of = =!9 Tris* succinate at p+ 2 <( = =69 potassiu' chloride( and = =!<9 'agnesiu' acetate The general procedure of )irenberg -!>6%. was e'ployed 6ashed cells were passed through a ;rench press at !6 === psi and then centrifuged at %= ===g for %='in to re'ove unbro:en cells and debris The supernatant fluid was dialy&ed against !== volu'es of the sa'e buffer for $hr the resulting preparation is designated the Qen&y'eR The ! = 'l reaction 'i,ture used for 'easure'ent of G !<CHleucine incorporation contained the indicated buffer -= !9 at p+ 2 <. and( in 'oles( 'agnesiu' acetate( 6=B potassiu' chloride( "=( 'anganous chloride( /B AT1( !=B 4T1( "B CT1( = !B phosphoenolpyruvate <B in g( piruvic Kinase( "=B all the a'ino acids e,cept leucine( "= Then content of radioactive leucine -specific activity !2 %" 'curiesJ''ole. was %$''oles -/=" === cp'. The reaction tubes were preincubation for % 'in at %2K and then en&y'e containing % $'g of protein -Lowry et al ( !>"!. was added( and the tubes were the sa'e e,cept that the preincubation and incubation were at =K The reaction was stopped by adding " 'l of "F TCA 1recipitated proteins were washed free fro' unincorporated a'ino acid as follows: the TCA*treated tubes were heated at >=K for /='in( then cooled and centrifuged The clear supernatant fluid was discarded and the precipitate was washed with another " 'l of "F TCA and then with " 'l of a %:! 'i,ture of alcohol and ether The washed precipitate was resuspended in "F TCA( decanted onto a 9illipore filter( and washed another five ti'es " 'l lots of "F TCA The filter containing the washed precipitate was dried in an e'pty scintillation vial for !" 'in at !==K ;inally !" 'l of scintillation fluid G= "F / "*diphenyla,a&ole and = =%F ! <*bis*/*-<*'ethyl*"*phenylo,a&oly.ben&ene in TolueneH was put in the vials( and the radioactivity was deter'ined in a li3uid scintillation spectro'eter The results with the different buffers are presented in Table 888 Both T@S and +@1@S were significantly better than Tris*succinate -in other e,peri'ents it was shown that Tris*succinate and Tris*+CL gave identical results. 1hosphate was appreciably worse 8t should be e'phasi&ed that in all of these instances the en&y'e had a long e,posure to Tris*succinate during its preparation and dialysis Therefore it is not unreasonable to suppose that T@S and +@1@S 'ight have given better protein synthesis rates if they had been used fro' the beginning of the preparation D8SC7SS85) There are two aspects to the evaluation of buffersSthe 3uestion of their buffering capacities in the desired p+ range and the entirely independent 3uestion of their side effects @valuation of the buffering capacity of any buffer poses no proble' if its pKa( its concentration( and the operating p+ are KnowB the ade3uacy of a buffer( as a buffer( depends only on the per'issible concentration of the buffer and the nearness of the operating p+ to the pKa 7nfortunately( not all biologists are aware of the very significant effects of te'perature on the pKa values of 'any buffers Thus( too often the wor:er who ad?usts a Tris buffer to p+ 2 $ at roo' te'perature is unaware that he has

prepared his 'aterial in the cold*roo' at p+ $ < and assayed its activity in a %2K incubation at p+ 2 < @valuation of the side effects of buffer is a 'uch 'ore difficult tas: since these side effects 'ay be different in every syste' studied 9oreover( differences in side effects can be obscured by differences in buffering efficiency 6e 'ust also consider that every buffer consist of two 'olecular species( protonated and nonprotinated( and these 'ay have entirely different side effects ;ortunately it is possible to ac3uire considerable infor'ation about the nonbuffering effects of buffers by testing the substance in syste's where there is little or no acid produced or consu'ed( or in syste's where there is already ade3uate buffering( or in syste's not very sensitive to p+ change A'ong our test the +ill reaction -as we 'easure it. is rather indifferent to p+( and the 'itochondrial syste' o,idi&ing succinate is already buffered with phosphate for phosphorylation Since these two tests 'easure pri'arily side effects( the evaluations 'ade in this paper often do not consider the p+*stabili&ing capacity of the buffer Thus when we say that +@1@S is 'ar:edly superior to Tris we are not fully recogni&ing the additional superiority conferred by its better buffering action near neutrality -@'phasis on testing the side effects of buffers has led us into so'e absurdities: it is ludicrous to co'pare Tris with 9@S as we have done( since their buffering ranges do not overlap and they could never be considered as alternatives 9@S should have been co'pared with 'aleate( di'ethylglutarate( histidine( and citrate . The criteria of suitability of buffers outlined in the introduction 'ust be realistic since al'ost all of the buffers tested which confor' to these criteria see' superior to the general applicability of our criteria opens the way to the preparation of al'ost any nu'ber of other substances having good buffer characteristics Syntheses li:e those described above are well within the ingenuity of bioche'ists( and we hope that an ever* widening assort'ent of buffers( tailored to specific applications( will be introduced by bioche'ists as needs arise ACK)56L@D49@)T The authors which to than: Dr 1hyllis 9 Bishop for her assistance in the early phase of this study

9agnesiu' co'pounds li:e 9gC5% nad 9g-5+./ act as antacids when ta:en in s'all doses( but in larger doses they act as la,atives A person suffering fro' a severe case of acid indigestion 'ight be unpleasantly surprised if she consu'es large a'ounts of one of these co'pounds to alleviate the proble'T The apparent change in 'edicinal properties is caused by the 'agnesiu' ion 6hen these co'pounds are used as antacids( 'agnesiu' is a QspectatorR ionB it is only along for the ride The actual neutrali&ation of acid is carried out by the anion portion of the salt The la,ative effect co'es about because 'agnesiu' ions in the large intestine creates a hypertonic condition The resulting os'otic pressure causes water to flow fro' the neighboring tissues into the large intestine( diluting the stool and causing diarrhea ;or this reason( over*the*counter drugs li:e 'il: of 'agnesia -a suspension of 'agnesiu' hydro,ide and water. have printed on their labels both the antacid dosage and the la,ative dosage YOUR (ERS(EC)I*E+ you want to re3uest that the 7 S ;ood and Drug Ad'inistration include 'andatory labels on 'agnesiu'*containing antacids to warn of their possible la,ative effects 6rite a letter to the ;DA( e,plaining the i'portance of this re3uest ,EY CONCE()S Se&eral general terms can -e use" to "escr!-e concentrat!on+ dilute( concentrated saturated( unsaturated( and supersaturated Un!ts o. concentrat!on t#at "eterm!ne prec!sel/ t#e num-er o. solute part!cles !n a spec!.!c amount o. sol&ent are 'olarity( weightJvolu'e percent( 'illigra' percent( parts per 'illion( and parts billion @3uivalents and 'illie3uivalents describe that a'ount of ionic charge on a solution Coll!gat!&e propert!es are t#ose propert!es o. a solut!on t#at "epen" onl/ on t#e num-er o. part!cles !n t#e solut!on0 not on t#e nature o. t#e part!cle' These properties include raising the boiling point and lowering the free&ing point of the solvent( and os'otic pressure Osmos!s !s t#e .lo$ o. $ater t#roug# a mem-rane .rom a reg!on o. lo$er solute concentrat!on to a reg!on o. #!g#er solute concentrat!on' 5s'otic pressure is the pressure that 'ust be applied to prevent such a flow 5s'olarity describes the total concentration of all particles in a solution 8sotonic solutions are ones with the sa'e os'olarity A hypotonic solution( isotonic has fewer solute particles and a hypertonic solution 'ore solute particles than a given solution ACIDS AND BASES AN)ACID

ob Knight was a 'iddle*aged insurance agent with a very sensitive sto'ach +e :ept a roll of antacids handy in his poc:et to control the acid indigestion he constantly suffered( and dran: large a'ounts of 'il: to :eep an ulcer fro' for'ing 5ne afternoon( Bob began suffering so'e very unusual sy'pto's +e beca'e e,tre'ely di&&y( had high and irregular blood pressure( felt nauseated( and had an acid

sto'ach that was worse than usual +is doctor sent Bob to the hospital for a series of test but was worse than usual +is doctor sent Bob to the hospital for a series of test but was unable to deter'ined what was wrong The sy'pto's soon disappeared( and Bob went ho'e But not after he returned to wor:( the sy'pto's reappeared Again he was hospitalised( and again the doctors could find nothing wrong 5n BobUs third visit to the hospital( his doctor even called in a psychiatrist for consultation The brea:through ca'e when a very deter'ined intern( after long discussion with Bob about his daily habits( found a reference to a condition called the Q'il:*al:aliR syndro'e in a 'edical te,tboo: The syndro'e( which included the sy'pto's suffered by Bob( is cased by a high inta:e of calciu'*containing antacids and large 3uantities of 'il: To understand what was happening to Bob( we 'ust ta:e a closer loo: at the wor: done by the sto'ach Cells in our sto'ach lining secrete hydrochloric acid -+CL.( which is very corrosive )or'ally the sto'ach lining secretes about / liters of +CL solution over a /<*hour period( 'ost co'ing after 'eals The proteins in our food( and drugs li:e alcohol and caffeine( sti'ulate the production of +CL 5ur e'otions also affect the secretion of sto'ach acid 8n the sto'ach this acid wor:s to :ill bacteria in foods( to soften foods( and the convert the inactive en&y'e pepsinogen into its active for'( pepsin( to begin the digestion of protein +ydrochloric acid is secreted into the sto'ach fluid in the for' of hydroniu' ions -+%5I( or hydrated protons. and chloride ions 8t is the presence of the hydroniu' ion that 'a:es the sto'ach contents acidic A ?elly*li:e layer of 'ucous lines the sto'ach to protect it fro' the acid The esophagus( however( does not have such a 'ucous 'e'brane 8f the sto'achUs contents bac: up into the esophagus( the result is QheartburnRSa stinging sensation behind the breastbone Swallowing one of the various products sold as antacids can neutrali&e the sto'achUs contents and the reduce the occurrence of heart*burn The Qactive ingredientR in the antacid tablets Bob was ta:ing is calciu' carbonate -CaC5%. Dissolved in the water 'i,ture in the sto'ach( calciu' carbonate dissociates to fro' the calciu' ion and the carbonate ion CaC5%-s.
+ 5 /

Ca/I -a3. I C5%/* -a3.

Calciu' Carbonate is an e,cellent antacid for 'any reasons: it acts rapidly( can neutrali&e a large a'ount of acid( and has a long*lasting effect at low cost As Bob discovered( however( it can also have serious side effects when used in large a'ounts over long periods 8ngesting too 'uch calciu' can lead to constipation and 'any cause Qacid rebound(R a condition in which the sto'ach produces even 'ore acid and 'ore heartburn An even greater danger( however( is caused by the high level of calciu' ions that can occur in the blood when a person repeatedly ta:es calciu'*based antacids The possibility of high blood calciu' concentration is significantly increased in persons who also drin: large a'ounts of calciu' concentration in significantly increased in persons who also drin: a'ounts of calciu'*rich 'il: or who have :idney proble's Although calciu' levels disrupt reactions in the body and cause the unusual sy'pto's suffered by Bob Such elevated calciu' levels can also i'pair :idney function and lead to the for'ation of :idney stones BobUs sy'pto's cleared up about a wee: after he reduced his inta:e of 'il: and switched to an alu'iniu'*'agnesiu' antacid to relieve his indigestion A critical balance between acids and bases 'ust be 'aintained in all parts of the living organis' to per'it nor'al physiological reactions to occur 8n this chapter we discuss

the nature of acids and bases and the ways in which their concentrations are regulated in the hu'an body ACIDS AND BASES+ )HE BRONS)ED1LO2RY DEFINI)ION Acids and bases play i'portant roles in living organis's Strong acids and bases can be 3uite har'fulB they da'age tissue by destroying protein 'aterial and drawing out water ;or e,a'ple( concentrated sulphuric acid is a strong dehydrating -water*re'oving. agent that rapidly in?ures tissues on contact Concentrated bases react whit the fast that 'a:e up the protective 'e'branes of cells( destroying such 'e'branes and causing even 'ore widespread destruction to tissues than acids do Strong laundry soaps and detergents contain bases Clothes containing wool and sil: -which are ani'al proteins. cannot be washed in such soaps because the base in the soap causes the fibers of these 'aterials to shrin: and partially dissolve Ac!"s are co'pounds that( when dissolved in water( produce solutions that taste sour( turn lit'us fro' blue to red( and react with 'etals such as &inc or 'agnesiu' to produce hydrogen gas Bases for' solutions that taste bitter( feel slippery to the touch( and therefore( is one in which either an acidic or basic solution is converted to neutral solution The reaction between( for e,a'ple( hydrochloric acid and sodiu' hydro,ide occurs between a3ueous ions The co'plete ionic e3uation for the reaction is

The net ionic e3uation for this reaction is

@MA91L@ !!*! ! 6rite the balanced e3uation for the neutrali&ation of sulphuric acid by potassiu' hydro,ide The proble' is as:ing us to write the correct for'ulas for the reactants of the neutrali&ation reaction( then to predict the products of that reaction( and finally to balance the e3uation for the reaction between sulfuric acid and potassiu' hydro,ide 6rite the correct for'ulas for the reactants Sulfuric Acid: +/S5< 1otassiu' hydro,ide: K5+ The products of is neutrali&ation reaction are and the salt potassiu' sulfate 6ater: +/5 1otassiu' sulfate K/S5<

)HE (# SCALE S'all changes in hydrogen ion concentration can be of great i'portance to living cells( and are critical in 'any fields of scientific investigation As a result( scientist are constantly 'easuring hydrogen ion concentrations 8n !>=>B a Danish bioche'ist na'ed SVren SVrenson developed the p+ scale to provide a way of showing the hydrogen ion concentration 'ore conveniently than by using a negative e,ponent -!=*2. or a deci'al

fraction -= ======!. p+ is the negative power to which the nu'ber != 'ust be raised to e,press the concentration of hydrogen ions in 'oles per liter 9athe'atically( G+IH = ! W !=*p+ or p+ = * Log G+IH At roo' te'perature( the G+ IH in pure water is ! W != *2 The p+ of pure water is therefore 2 Because in pure water the G+ IH e3uals G5+*H( pure water is considered to be neutral This 'eans that the p+ of a neutral solutions is 2 Acidic solutions are ones in which the hydrogen ion concentration is greater than the hydro,ide ion concentration ;ro' table !! % we see that for an acidic solution is less than 2 A basic solution is one in which the hydrogen ion concentration is less than the hydro,ide ion concentration( and the p+ is greater than 2 -;ig !! !. The 'easure'ent of p+ is an i'portant laboratory procedure because the p+ of a solution affects the activity of biological 'olecules This 'eans that ph can influence the behaviour of cells and even entire organis's ;or e,a'ple( bacteria grow best in a very s'all range of p+ The ph of culture 'edia 'ust therefore be carefully controlled @n&y'es( the biological catalysts( wor: best in narrow li'its of p+ that can vary fro' an opti'u' p+ range of ! to < for pepsin -an en&y'e in the sto'ach. to an opti'u' p+ range of $ to > for trypsin -an en&y'e in the s'all intestine. 9ost body fluids are 'aintained in a very narrow range of p+ that( if changed( can be to,ic to the organis' -Table < <.